WO2010075372A9

WO2010075372A9 - Inhibitors of mre11, rad50 and/or nbs1

Info

Publication number: WO2010075372A9
Application number: PCT/US2009/069171
Authority: WO
Inventors: Jean Gautier; Levy Kopelovich
Original assignee: The Trustees Of Columbia University In The City Of New York; The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2008-12-22
Filing date: 2009-12-22
Publication date: 2010-09-02
Also published as: WO2010075372A1

Abstract

The invention provides, inter alia, compositions comprising an inhibitor of MRE11, RAD50 and/or NBSl and methods of using such inhibitor for modulating the cellular response to DNA damage, arresting the cell cycle, and/or treatment of cancer. The inhibitor is, e.g., a compound of formula I:

Description

INHIBITORS OF MREIl, RAD50 AND/OR NBSl

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/203,377, filed December 22, 2008, which is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0001] The invention described herein was made with government support under contract numbers NOl-CN-25110 awarded by the National Cancer Institute, and CA95866 and CA92245 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to inhibitors of MREIl, RAD50, and/or NBSl, genes involved in signaling DNA damage and assembling damaged DNA into complexes that induce a phosphorylation cascade leading to checkpoints in the cell cycle that can arrest cell division.

BACKGROUND OF THE INVENTION

[0003] Cells are constantly exposed to endogenous and exogenous DNA damage. However, double-strand breaks (DSBs) in DNA are the most harmful. When unchecked, DSBs may lead to mutations, chromosomal aberrations and eventually to the development of cancer (Kastan & Bartek, Nature 432(7015): 316-323 (2004); Sancar et al. Annu Rev Biochem 73: 39- 85 (2004)). DSBs result from disruption of the phosphodiester backbone on both strands of the DNA molecule with a geometry that prevents end juxtaposition by either base pairing or chromatin structure cohesion. These lesions pose a considerable threat to genomic stability because the DNA can lose information on both strands as well as its physical connection to the centromere.

[0004] Cells respond to DNA damage by activating a multi-faceted response called the DNA damage response (Hensey & Gautier, Prog. Cell Cycle Res. 1: 149-162 (1995); Zhou & Elledge, Nature 408 (6811): 433-39 (2000)). The DNA damage response involves a network of signal transduction pathways that monitor and sense aberrant DNA and chromosome structures, and then coordinately trigger checkpoint pathways that prevent cell cycle progression and activate DNA repair systems. These signaling pathways operate throughout the cell cycle. Activation of a DNA damage checkpoint can prevent cellular replication by inducing delays in the Gl, S, or G2 phases of the cell cycle.

[0005] Thus, while damage to DNA and chromatin structure can give rise to mutations and chromosomal translocations that may cause cell tumors and developmental problems, there are situations where a failure to repair DNA damage can have beneficial effects. For example, when DNA damage triggers a checkpoint pathway in cancer cells, those cancer cells may not replicate, or may only replicate slowly. Hence, agents that can manipulate the cellular response to DNA damage are useful agents for modulating the cell cycle. Similarly, agents that cause DNA damage, or that prevent cells from repairing DNA damage, may prevent undesirable cells (e.g., cancer cells) from multiplying.

SUMMARY

[0006] The invention provides, inter alia, inhibitors of MRN (MREl 1-RAD50-NBS1), which is a complex involved in sensing DNA damage and maintaining genome stability during DNA replication. MRN is essential for cell survival. As described herein, inhibitors of MRN can arrest exponentially dividing cells at the G2/M phase of the cell cycle and can therefore be used for cancer treatment. In addition, according to the invention, many cancer cells have one or more defects in the pathways relating to maintaining genomic stability and repairing DNA. By inhibiting the MRN pathway, the present inhibitors and methods can, e.g., inhibit cancer cell growth and sensitize the cells to chemotherapeutic agents and/or radiation.

[0007] One aspect of the invention is a composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I:

wherein:

A is a heterocyclyl which is attached to L via a ring carbon of a ring consisting of carbon and one to three Yn where n is 1, and which has 0-2 double bonds between ring carbons; L is a bond or a linker selected from C₁-C₄ alkylene, C₁-C₄ alkenylene, C₂-C₄ alkenylene;

B is selected from the group consisting of phenyl, ortho-fused bicyclic carbocyclic, and heteroaryl, wherein the ortho-fused bicyclic carbocyclic has nine or ten ring atoms and in which at least one ring is aromatic, and wherein the heteroaryl is a radical attached to L via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and from one to four Y_n where n is 1, or a radical attached to A via a ring carbon of an ortho-fused bicyclic heterocycle consisting of eight to ten ring atoms wherein one of the rings is an aromatic ring containing five or six ring atoms consisting of carbon and from one to four Y_n where n=l;

X is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(P), wherein P is absent or is H;

Y is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(Q), wherein Q is absent or is H, O, (C₁-C₄)alkyl, phenyl or benzyl;

Z is hydroxy or amine; and n is 0 or 1, provided that ring A has at least one Y group; and

— is an optional double bond; or an enantiomer, optical isomer, diastereomer, or pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the compound can have formula II:

wherein X, Y and Z are as defined herein.

[0009] In other embodiments, the compound can be a compound called "Mirin," which has the following structure:

[0010] Other chemotherapeutic agents can be included in the compositions of the invention.

[0011] Another aspect of the invention is a method of treating cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula I or II. In some embodiments, the compound is Mirin, with the following structure:

[0012] According to the invention, the compound may in some instances be administered with a chemotherapeutic agent. For example, the chemotherapeutic agent can be an anti-microtubule agent, platinum coordination complex, alkylating agent, antibiotic, topoisomerase II inhibitor, antimetabolite, topoisomerase I inhibitor, hormone, signal transduction pathway inhibitor, non-receptor tyrosine kinase angiogenesis inhibitor, immunotherapeutic agent, proapoptotic agent, cell cycle signaling inhibitor or a combination thereof.

[0013] The compound employed in the therapeutic methods of the invention can, for example, inhibit MEREIl activity. In some embodiments, this method inhibits cancer or tumor cell growth. In other embodiments, this method sensitizes cancer cells to chemotherapy or radiation therapy.

[0014] In another aspect of the invention, a method of inhibiting MREIl activity is provided comprising administering to a mammal a therapeutically effective amount of a compound of formula I or II or of Mirin. [0015] A further aspect of the present invention is a method of sensitizing cancer cells to chemotherapy or radiation therapy comprising administering to a mammal a therapeutically effective amount of a compound of formula I or II or of Mirin.

[0016] Another aspect of the invention is a method of identifying an anticancer agent comprising: (a) contacting an assay mixture with a test agent, wherein the assay mixture comprises a H2AX peptide having the amino acid sequence of SEQ ID NO:2, ATP, DNA with double- stranded breaks, and an inhibitor of MREIl, RAD50, NBSl, or a combination thereof; (b) observing whether phosphorylation of the peptide (SEQ ID NO:2) increases relative to a control assay lacking the test agent; and (c) identifying the test agent as an anticancer agent when the phosphorylation of the peptide (SEQ ID NO:2) increases.

[0017] Another aspect of the invention is a method of identifying an agent that can modulate a pathway involving MREIl, RAD50, NBSl, and/or ATM comprising: (a) contacting an assay mixture with a test agent, wherein the assay mixture comprises a peptide having the amino acid sequence of SEQ ID NO:2, ATP, DNA with double- stranded breaks, and an inhibitor of MREIl, RAD50, NBSl, or a combination thereof; (b) determining whether phosphorylation of the polypeptide (SEQ ID NO:2) increases or decreases relative to a control assay that does not contain the test agent; and (c) identifying an agent that modulates the MRN- ATM pathway when that agent increases or decreases the phosphorylation of the polypeptide (SEQ ID NO:2) relative to the control assay. In addition to the ingredients listed, the assay mixture can further comprise a cytoplasmic extract and/or calcium. Such a cytoplasmic extract can be a Xenopus oocyte cytoplasmic extract. In some embodiments, the ATP has a detectable label on its terminal (γ) phosphate. Moreover, each assay can be performed in a well of a 96- well plate with V-shaped wells. When using such a 96- well plate, each assay can be performed in an assay volume of about 1-5 microliters. After the phosphorylation reaction has progressed, the assay mixtures can, for example, be transferred to P81 phosphocellulose coated 96-well plates, where the H2AX (SEQ ID NO:2) peptide binds to the P81 phosphocellulose. In some embodiments, phosphorylation of the peptide (SEQ ID NO:2) is detected by detecting a labeled phosphate linked to the peptide. In other embodiments, the phosphorylation of the peptide (SEQ ID NO:2) is detected using an antibody that detects phosphorylated peptide (SEQ ID NO:2) but substantially no non-phosphorylated H2AX (SEQ ID NO:2) peptide.

[0018] Another aspect of the invention is a method of identifying an anticancer agent comprising: (a) contacting an assay mixture with a test agent, wherein the assay mixture comprises cancer cells and an inhibitor of MREI l, RAD50, NBSl or a combination thereof; (b) observing whether growth of the cancer cells in step (a) changes; and (c) identifying the test agent as an anticancer agent if the cancer cell growth decreases. For example, the inhibitor can be Mirin or a compound of formula I or II. In other embodiments, the inhibitor is a nucleic acid that inhibits the expression or translation of MREIl, RAD50, NBSl, or a combination thereof. Such a nucleic acid can be homologous or complementary to about 10 to about 50 nucleotides of SEQ ID NO:3, 4 or 5, or have any one of SEQ ID NO:6-17, or a combination thereof.

[0019] Another aspect of the invention is a method of inhibiting MRN (MREIl- RAD50-NBS Independent activation of ATM (Ataxia- Telangiectasia Mutated) comprising contacting a cell capable of expressing the MRN (MREl 1-RAD50-NBS1) with at least one nucleic acid that inhibits the expression or translation of MREIl, RAD50, NBSl, or a combination thereof. In some embodiments, the nucleic acid is about 10-50 nucleotides in length, or has any number of nucleotides from about 10 to about 50 nucleotides. For example, the nucleic acid can be homologous or complementary to about 10 to about 50 nucleotides of SEQ ID NO:3, 4, or 5. Specific, non-limiting, examples of nucleic acids that inhibit the expression or translation of MREIl, RAD50, NBSl include any one of SEQ ID NO:6-17, or a combination thereof. In some embodiments, the cell can also be contacted with a compound of formula I or II, or the Mirin compound. While the cell can be in an in vitro environment, the cell can also be within a mammal. This method can also involve administering to the mammal a chemotherapeutic agent.

[0020] Another aspect of the invention is a method of inhibiting MREIl activity comprising contacting the MREIl with a compound of the following structure (Mirin):

to thereby inhibit MREl 1 activity. A further aspect of the invention is a method of inhibiting MRN (MREl 1-RAD50-NBS l)-dependent activation of ATM (Ataxia-Telangiectasia Mutated) comprising contacting MRN with Mirin. In some embodiments, the method is performed in vitro. In other embodiments, the method is performed in a living cell or in a mammal. DESCRIPTION OF THE FIGURES

[0003] FIGS. IA - E illustrate that Z-5-(4-Hydroxyphenylidene)-2-imino-1,3- thiazolidin-4-one (Mirin) is an inhibitor of ATM activation by double-stranded breaks in DNA (DSBs) in Xenopus extracts.

[0004] FIG. IA shows the screening protocol. Xenopus extracts were treated with DSB- containing DNA. Aliquots of extracts were then transferred to 96-well plates containing 80 compounds (columns 2-11) per plate. Positions A 1, A12, B1, B 12, C1 and C12 were used for positive controls (PC; no compound). Caffeine, an ATM inhibitor, was used as a control for inhibition in positions D1, D12, E1, and E12. Positions F1, F12, G1, G12, H1, and H12 were used as negative controls (NC) with non-phosphorylatable peptide (AVGKKAAQAAQEY, SEQ ID NO:1). ATM activity was assayed by measuring the incorporation αf 3™-ATP into a peptide derived from histone H2AX peptide (AVGKKASQASQEY, SEQ ID NO:2). The reactions were transferred to a 96-well P81 phosphocellulose plate, washed, dried, and exposed to a Phosphorlmager screen.

[0005] FTG. IB shows the Z' values calculated for the H2AX peptide phosphorylation assay screen (125 plates). The 2' value for each plate was calculated following the formula: 1- ((standard deviation positive sample values + standard deviation negative sample values) / (average of positive sample values -average negative sample values)). As shown, 90 % of 125 plates had a Z' factor value of 0.3 or above. The average Z' factor was 0.57 indicating a robust assay. Mirin was identified from plate # 68 with a Z' factor of 0.87. Z' values below 0.2 are indicated with squares [■]. These plates were not included for the calculation above.

[0006] FIG. 1C shows the structure of Z-5-(4-hydroxyphenylidene)-2-imino-1,3- thiazolidin-4-onc (Mirin), of Z-5-(4-hydroxybenzylidene)-2-thioxo-I ,3-imidazolidin-4-one and of 6-phenyl-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (5122443).

[0007] FIG. ID graphically illustrates percent inhibition of H2AX peptide phosphorylation by Mirin and 6-phenyl-2-thioxo-2,3-dihydro-4(1H)-pvrimidinone (5122443). Xenopus extracts were incubated with increasing concentrations of Mirin or 5122443 in the presence of DSB-containing DNA (5 ng/μl). ATM activity was assayed as described in (A) and percentages of inhibition were calculated as described in Example 1.

[0008] FIG. IE graphically illustrates the phosphorylation of the histone 112 AX peptide (AVGKKASQASQEY, SEQ ID NO:2) under different conditions. Mock-depleted extracts (lanes 1 to 3) or ATM-depleted extracts (lanes 4 and 5) were incubated with streptavidin-bound biotinylated DNA (DNA, lanes 2-3)' in the presence of 100 μM Mirin (lanes 3 and 5) or DMSO (lanes 1, 2 and 4). After DNA pull-down, ATM activity was assayed in soluble fractions by measuring H2AX peptide phosphorylation. The Western blot below shows the extent of ATM depletion.

[0009] FIGS. 2A - D illustrate that Minn inhibits MRN (MRE 11-RAD50-NB Sl)- dependent activation of ATM (Ataxia-Telangiectasia Mutated).

[0010] FIG. 2A shows that Mirin inhibits ATM-dependent phosphorylation of Nabs I and Chk2 in Xenopus extracts. Extracts were incubated with DMSO (lanes I and 2) or with 50 μM, 100 ,uM and 500 μM Minn (lanes 3-5) and then treated with DSB-containing DNA. NDSl (upper panel) and Chk2 (lower panel) electrophoretic mobility were monitored by Western blot using antibodies directed against NBSl and Chk2.

[0011] FlG. 2B shows that Mirin inhibits MRN-dependent activation of ATM in extracts. Mock- (dark grey, lanes 1 to 5) or MREl 1 -depleted extracts (light grey, lanes 6 to 9) were incubated with 100 μM of Mirin (lanes 3, 5, 7 and 9). ATM activation was triggered by addition of DSB-containing DNA at Lhe indicated concentrations. Following DNA pull-down, ATM activation was assayed in the resulting soluble fractions by either H2AX peptide phosphorylation or by Western blot using an antibody against phosphorylated S1981 of ATM (P-ATM). Each bar represents the average of four different experiments with the standard deviation shown. Values marked with asterisks * (3 and 5; 7 and 9) are significantly different (PO.003). MREl I depletion Is shown below the graph.

[0012] FIG. 2C Shows that Mirin prevents MKN- and DNA-dependent activation of dimeric ATM in vitro Dimeric ATM activity was assayed in the presence of DNA alone (lane 2), MKN alone (lane 3), MRN and DNA (lanes 4 to 7), as well as 10 VM or 50 μM of Mirin (lanes 5 and 6 respectively). ATM activation was monitored by Western analysis of Ser15- phosphorylated p53.

[0013] FlG. 2D shows that Mirin does not inhibit the activity of monomelic ATM in vitro. Monomelic ATM activity was assayed as in C in the presence of 10 μM or 50 μM of Mirin (lanes 2 and 3). ATM activity was monitored by western analysis of Ser15- phosphorylated p53.

[00141 FIGS, 3A - E illustrate that Mirin inhibits the nuclear activity of MREIl.

[0015] Fig. 3 A shows that Mirin does not trigger MRN complex dissociation in extracts. Extracts were Incubated with recombinant FLAG-tagged human MRN complex and treated with DMSO (lanes 1, 2, 5 and 6) or Mirin (50 or 100 μM, lanes 3, 4, 7, 8) for 15 min. Recombinant MRN was isolated using FLAG antibodies. Supernatants (lanes 2-4) and FLAG- resin (lanes 6-8) were processed for Western blot by probing with antibodies against FLAG, MREl 1 or RAD50. Xenopus RAD50 protein is not recognized by the RAD50 antibody. [0016] FIG. 3 B ahows that Mirin doβ9 not dissociate endogenous MREI l- associated complexes. 50 μl of extracts were treated with DMSO (upper panel) or 100 μM Mirin (lower panel) for 15 minutes then processed for Superose 6 gel chromatography. Each fraction was analyzed by Western blot using an antibody directed against MREI I. Elution positions of molecular weight markers for 667 kD and 440 kD are indicated on top.

[0017] FIG, 3C shows Mirin does not inhibit ATM and MREI l binding to DNA. Extracts were treated with DMSO (lanes I, 2, S and 6) or with 1 nM or 0.1 nM concentrations of Mirin (lanes 3, 4, 7, 8) prior to incubεtion with DSB-containing DNA (1.2 κ 10" ends/μl) (lanes IA and 6-8). Following DNA pull-down, phosphorylation of ATM on Serine 1981 (P- ATM, upper panel), ATM (middle panel) and MREIl (lower panel) were monitored by Western blot in resulting soluble-fractions (lanea 1-4, left panel) and in DNA-bound fractions (lanes 5-8, right panel).

[0018] FIG. 3D shows that Mirin does not inhibit MRN-associated DNA tethering activity, Mock-(dark grey, lanes 1 to 4) or MREIl -depleted extracts (light grey, lanes 5 to 7) were incubated with DMSO (lanes 1, 2 and 5) or 100 μM or 10 μM Mirin as indicated (lanes 3, 4, 6 and T). Extracts were then incubated with strcptavidin-bound DNA ( 1.2x10¹¹ ends/μl) and free radioactive DNA ( 1.2x10¹¹ ends/μl). DNA-tethering activity was assayed by measuring the radioactivity associated with streptavidin-bound DNA. Each bar represents an average of six independent experiments with the standard deviation shown. Values marked with asterisks are significantly different (P=O OOl),

[0019] FIG. 3E shows that Mirin inhibits the nuclease activity of MRN. Purified recombinant human MRN was incubated for 20 minutes with 5 '-labeled immobilized DNA in the presence of DMSO or 0.05, 0.1, 0.2, 0.4, or 0.8 mM Miriα. The radioactivity released In soluble fractions was determined. MRN-independent nuclease activity (assayed without MnCl₂) was subtracted prior to calculating the percentage of inhibition. The average of three experiments is shown. It was found that Mirin inhibits Mrel 1 nuclease activity at 100μM.

[0020] FIGS. 4A -E show that Mirin abolishes the G2/M checkpoint in the cell cycle.

[0021] FIG. 4A illustrates the cell cycle distribution of TOSA4 cells. TOSA4 cells were treated with DMSO (lane 11) or Mirin (Ianes 2-5), and cells were processed for FACS analysis. The cell cycle distribution (G1, S and G2/M) is shown in the plot of percent of cells in cell Cycle phases. Each bar represents an average of three independent experiments with the standard deviation shown.

[0022] FIG. 4B shows that a specific ATM inhibitor, KU-55933, does not abolish Mirin-induced G2/M arrest. Exponentially growing U2OS cells were treated with the indicated concentrations of 0 μM, 25 μM, or 100 μM Mirin and 0 μM or 10 μM KU-55933. After 24 hours, cells were harvested, fixed with 70% ethanol, stained with propidium iodide and procesied fur cell cycle analysis by FACS. The graphs plot FL2-Area vs. Counts.

[0023] FIG.4C shows that Mirin prevents ATM autophosphorylation on residue S 1981. U2OS cells synchronized in Gl were incubated 0 μM, 25 μM, or 100 μM Mirin and then Mock-Irradiated (0 Oy) or irradiated with 10 Cy. Cells wore harvested 30 minutes after irradiation and processed for Western blot with antibodies against phosphorylated S 1981 of ATM (top panel) αr total ATM (bottom panel).

[0024] FIG. 4D shows that Mirin abolishes the IR-induced G2/M checkpoint in UZOS cells. U2OS cells were synchronized in G2 (3ee Example I), treated with DMSO (Control) or 25 μM Mirin, irradiated (10 Gy) or not irradiated, and mitotic cells were trapped with nocodazole. The cells were then stained for a marker of mitotic cells, phosphorylated histone H3. A typical FACS profile fur each sample is shown.

[0025] FIG. 4E shows that treatment with Mirin resulted in a ten-fold increase in cells entering mitosis after irradiation. The plot illustrates the average percentage of phospho-H3- positive cella (in mitosis) following IR in control (DMSO) and 25 μM Minn-treated cells. The average of three independent experiments with standard deviation is shown.

[0026] FIGS. 5A - B show that Mirin inhibits homology-dependent DNA repair in human cells.

[0027] FIG. 5A illustrates the cytotoxicity of Mirin in HEK293 cells. HEK293 cells were treated for 24 hours with DMSO or 10 μM, 25μM, 50 μM, or 100 μM of Mirin. Cells were grown for 10 days, then fixed and stained with 10% Giemsa. Stained plates were counted for colonies. Percentage survival is expressed as the average number of colonies on a treated plate divided by the average number of colonies on the control plate (0 μM Minn). Each bar represents an average of three independent experiments with the standard deviation shown.

[0028] FIG. 5B illustrates a gene conversion assay. Top: schematic of the assay. Bottom; TOSA4 cells treated with DMSO or 10 μM, 25 μM, 50 μM, or 100 μM Mirin were transfectod with I-Seel expressing plasmid. After 24 hours, GFP-expressing cells were then scored by cell sorting. The average of four independent experiments is shown. Each bar represents an average of five independent experiments with the standard deviation shown,

[0029] FIGS. 6A - B illustrate how ATM activation in cell-free extracts can be monitored. Phosphorylation of a reporter peptide derived from H2AX was monitored following a 10-minute incubation of mock- or ATM-depleted membrane-free cytosol with DNA containing double-stranded breaks. ATM depletion was achieved using specific antibodies (Λbl, Ab2 and Ab3) or a CJ-tenninal NBSl peptide.

[0030] FIG. 6A shows the sequence of the ATM-binding domain of Xenopus NBSl (a 22 amino acid peptide sequence from the NBSl C-terminus, VREESLAEDLFRYNPKPSKRRR, SEQ ID NO:20). The sequence of a control peptide (VRAΛSLAAALFRYNPKPSKRRR, SEQ ID NO:21) is also shown.

[0031] FIG. 6B graphically illustrates the depletion efficiencies as determined by Western blot. ATM depletion resulted in the inhibition of H2AX peptide phosphorylation.

[0032] FIGS. 7A - B show activation of ATM under the assay conditions described below.

[0033] Fig. 7A schematically illustrates an assay for monitoring ATM activity.

MREH -depleted Xenopus oocyte extracts were incubated with immobilized DNA harboring double-stranded breaks (Step 1). The resulting soluble fractions (containing monomelic, inactive ATM) were then treated wrth buffer, MRN complex, NBSl C-terminal peptide (ATM-binding domain) or a control peptide,

[0034] FIG. 7B graphically illustrates activation of ATM by the indicated factors. It 3how3 that the ATM binding domain of Nbsl is needed to convert unphosphorylated ATM monomers into fully active monomers in the absence of DNA.

[0035] FIG. 8 illustrates a dose-response assay using a high throughput assay to detect which DSB-containing DNA concentrationa are optimal for identifying inhibitors and agonists of the MRN-ATM pathway. The section of a 96-well plate shown illustrates the intensity of H2AX peptide phosphorylation at doses of DSB-containing DNA ranging from 1 to 100 ng/μl. The boxed concentrations were chosen for screening for inhibitors (5 ng/μl) and agonists (2 ng/μl) of the MRN-ATM pathway.

[0036] FIG.9 shows the synthetic scheme of Mlrin.

DETAILEiD DESCRIPTION

[0037] The invention involves, inter alia, chemosensitizing agents that are useful anti- tumor agents. These agents can be used for sensitizing cancer cells to chemotherapeutic agents. For example, administration of the chemosensitizing agents of the invention can reduce the amount or dosage of chemotherapeutic agent(s) required for effective cancer treatment, thereby reducing the side effects of the chemotherapeutic agent (s), Compounds

[0038] One embodiment of the invention is a compound of formula I:

wherein:

A is a heterocyclyl which is attached to L via a ring carbon of a ring consisting of carbon and one to three Y_n where n is 1, and which has 0-2 double bonds between ring carbons;

L is a bond or a linker selected from C₁-C₄ alkylene, CrC₄ alkenylene, or C₂-C₄ alkynylene;

X is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur and N(P), wherein P is absent or is H;

Y is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(Q), wherein Q is absent or is H, O, (C₁-C₄)alkyl, phenyl, or benzyl;

—is an optional double bond; or an enantiomer, optical isomer, diastereomer, or pharmaceutically acceptable salt thereof.

[0057] Preferably, the B ring is an ortho-fused bicyclic heterocycle, which contains a benzyl as one of the fused rings. Also preferably, the bicyclic heterocycle is derived by fusing a propylene, trimethylene, or tetramethylene diradical to the five or six membered aromatic ring. [0058] Preferably, the A heterocyclyl is a 3-to 10-membered ring, more preferably a 3- to 8-membered ring. More preferably, the A heterocyclyl is a 3-to 7-membered ring or a 3-to 6- membered ring. In one alternative embodiment, L is a bond directly between a carbon atom in the A-ring and a carbon atom in the B ring.

[0059] Another embodiment is a compound of formula II:

wherein X, Y Z, and — are as defined herein.

[0060] One example of a compound useful in the compositions and methods of the invention is a compound with the following structure:

[0061] In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts of compounds of the present invention are organic acid addition salts formed with acids which form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and a- glycerophosphate. Suitable inorganic salts of the compounds of the present invention may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate and carbonate salts.

[0062] Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0063] It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine MRE inhibitory activity using the standard tests described herein, or using other similar tests which are well known in the art.

[0064] The compounds of the invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes. Further details on compositions and formulations containing the compounds of the invention are provided below. Definitions

[0065] The following definitions are used, unless otherwise indicated.

[0066] "Halo", "halogen", and "heteroatom" are used interchangeably herein, and mean fluoro, chloro, bromo, or iodo.

[0067] The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl- substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branched chains), and more preferably 20 or fewer, such as from 1 to 8. In the present invention, when a range is recited all values within the range, including the end points are within the scope of the present invention, including all sub-ranges that may be formed or identified within the stated range. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6, or 7 carbons in the ring structure.

[0068] As noted above, alkyl denotes both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to. [0069] The term "alkenylene" means a divalent group derived from a straight or branched chain hydrocarbon containing at least one double bond. Representative examples of alkenylene include, but are not limited to, =CH-, =CH-CH₂-, and =CH-CH₂-CH₂-, and =CH-

CH₂- CH₂- CH₂- .

[0070] The term "alkylene" means a divalent group derived from a straight chain hydrocarbon containing only single bonds. Representative examples of alkylene include, but are not limited to, -CH₂-, -CH₂CH₂-, -CH₂-CH₂-CH₂-, and -CH₂-CH₂-CH₂-CH₂.

[0071] The term "alkynylene" means a divalent group derived from a straight or branched chain hydrocarbon containing at least one triple bond. Representative examples of alkynylene include, but are not limited to, -C≡C-, -C≡C-CH₂-, and -CH₂-C≡C-CH₂-.

[0072] The terms "amine" and "amino" are ad-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by

wherein R', R", and R'" each independently represent a hydrogen or a hydrocarbyl group, or R' and R" taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.

[0073] The term "aryl" as used herein includes substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 3- to 8- membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like. In formula I, ring B may be aryl which can be a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.

[0074] The terms "carbocycle", "carbocyclyl", and "carbocyclic", as used herein, refer to a non-aromatic saturated or unsaturated ring in which each atom of the ring is carbon. Preferably, a carbocycle ring contains from 3 to 10 atoms, more preferably from 3 to 8 atoms, including 5 to 7 atoms, such as for example, 6 atoms.

[0075] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic single ring structures, preferably 3-to 8-membered rings, more preferably 5-to 7- membered rings, even more preferably 5-to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like. In formula I, ring B may be a heteroaryl which can encompass a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four Y, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom.

[0076] The terms "heterocyclyl", "heterocycle", and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 8-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0077] The term "hydrocarbyl", as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon- hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, and alkynyl.

[0078] Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.

[0079] Specific embodiments include that (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C₃-C₆)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), or quinolyl (or its N-oxide).

[0080] As used herein the term "animal" refers to mammals, preferably mammals such as humans. Likewise, a "patient" or "subject" to be treated by the method of the invention can mean either a human or non-human animal, preferably a human.

[0081] As used herein, the term "cancer" refers to any neoplastic disorder, including such cellular disorders as, for example, renal cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer, mammary adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, and gastrointestinal or stomach cancer. Preferably, the cancer which is treated in the present invention is melanoma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, or ovarian cancer.

[0082] The "growth state" of a cell refers to the rate of proliferation of the cell and the state of differentiation of the cell

[0083] As used herein, "hyper-proliferative disease" or "hyper-proliferative disorder" refers to any disorder which is caused by or is manifested by unwanted proliferation of cells in a patient. Hyper-proliferative disorders include but are not limited to cancer, psoriasis, rheumatoid arthritis, lamellar ichthyosis, epidermolytic hyperkeratosis, restenosis, endometriosis, and abnormal wound healing.

[0084] As used herein, "proliferating" and "proliferation" refer to cells undergoing mitosis.

[0085] As used herein, "unwanted proliferation" means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention. A pathological state that ensues because of the unwanted proliferation of cells is referred herein as a "hyper- proliferative disease" or "hyper- proliferative disorder".

[0086] As used herein, "transformed cells" refers to cells that have spontaneously converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. For purposes of this invention, the terms "transformed phenotype of malignant mammalian cells" and "transformed phenotype" are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological, or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals.

Inventive Compounds are Inhibitors of the Mrell-Rad50-Nbsl complex

[0087] The MRN (MREl 1-RAD50-NBS I)-ATM (Ataxia-Telangiectasia Mutated) pathway is essential for sensing DNA double- stranded breaks and effectively responding thereto. Cells respond to DNA damage by coordinating DNA repair with cell cycle progression and/or apoptosis. This response is essential to maintain the integrity of the genome. Inherited mutations in the ATM, Mrell and Nbsl genes are responsible for the cancer-prone syndromes Ataxia- Telangiectasia (A-T), Ataxia Telangiectasia-Like Disorder (ATLD) and Nijmegen Breakage Syndrome (NBS), respectively. These syndromes share clinical and cellular phenotypes including hypersensitivity to ionizing radiation (IR), radio-resistant DNA synthesis, checkpoint deficiencies and chromosomal instability, chromosomal breaks in particular.

[0088] ATM belongs to the phosphatidylinositol-3' kinase-related kinase (PIKK) family and plays a critical role in the maintenance of genome integrity (Lavin et al., Mutat Res 569, 123-32(2 005)). In response to agents that generate DSBs, inactive dimeric ATM protein dissociates into phosphorylated monomers. The MRN complex is critical for this activation process. In addition to phosphorylating ATM, the MRN complex also recruits ATM to damaged DNA (You et al., MoI Cell Biol 25, 5363-79 (2005); Lee et al., Science 304, 93-6 (2004); Lee et al., Science 308, 551-4 (2005); Carson et al., Embo. J 22, 6610-20 (2003); Difilippantonio et al., Nat Cell Biol 7, 675-85 (2005); Dupre et al., Nat Struct MoI Biol 13, 451- 7 (2006); Kozlov et al., Embo J 25, 3504-14 (2006); Gatei et al., Nat Genet 25, 115-9 (2000); Paull et al., Cell Cycle 4, 737- 40 (2005); Falck et al., Nature 434, 605-11 (2005); and Uziel et al., Embo J 22, 5612-21 (2003)). Upon activation, ATM phosphorylates several substrates, including p53, CHK2, NBSl and BRCAl, that together coordinate cell cycle arrest and DNA repair (Shiloh et al., Nat Rev Cancer 3, 155-68 (2003)).

[0089] The MRN complex displays DNA binding and tethering activities and functions as both a single- and double-stranded DNA endonuclease as well as a 3'-5' double-stranded exonuclease (D'Amours et al., Nat Rev MoI Cell Biol 3, 317-27 (2002); Paull et al., MoI Cell 1, 969-79 (1998)). MRN is required for the maintenance of genome stability during DNA replication, for telomere length maintenance, and for DNA repair (Haber et al., Cell 95, 583-6 (1998)). As a result, the Mrell, Rad50, and Nbs 1 genes are each essential for cell viability whereas ATM is not.

[0090] The inventors and co-workers have designed a screen to identify small molecules with potential radio- and/or chemo-sensitizing properties (Kasten & Bartek, Nature 432, 316-23 (2004); Zhou & Bartek, Nat Rev Cancer 4, 216-25 (2004)) that inhibit the MRN-ATM pathway in response to DSBs. The screen was performed in cell-free extracts derived from Xenopus eggs that recapitulate many aspects of the MRN- and ATM-dependent responses to DSBs as well as some of the aberrant phenotypes, such as radio-resistant DNA synthesis (RDS), observed in cancer prone syndromes (Dupre et al., Nat Struct MoI Biol 13, 451-7 (2006); Costanzo et al., MoI Cell 6, 649-59 (2000); Costanzo et al., MoI Cell 8, 137-47 (2001)). Xenopus extracts have been used previously to identify small molecule inhibitors of actin assembly, spindle assembly and cell cycle progression (Peterson et al., Proc Natl Acad Sci U S A 98, 10624-9 (2001); Verma et al., Science 306, 117-20 (2004); Wignall et al., Chem Biol 11, 135-46 (2004)). A cell free screening assay for identifying effectors of Fanconi anemia pathway activation using Xenopus egg cell free extracts is disclosed in W02007/067261. In our screen, the DNA damage response was triggered by incubating cell-free extracts with Haelll- digested plasmid DNA which mimics DNA DSBs. The read-out assay for ATM activity was phosphorylation of a peptide (AVGKKASQASQEY, SEQ ID NO:2) derived from histone H2AX (Costanzo et al., MoI Cell 6, 649-59 (2000); Costanzo et al., PLoS Biol 2, EIlO (2004)) carried out in a 96-well plate format (see, FIG. IA, FIG. 8, and Example 1).

[0091] In this forward chemical genetic screen, a small molecule can target any step in the pathway leading to H2AX phosphorylation. As described herein, a 10,000- compound "DiverSet" library from Chembridge Corporation was used that had previously led to the identification of small molecules that inhibit p53 activity or interfere with mitosis and spindle dynamics (Komarov et al., Science 285, 1733-7 (1999); Mayer et al., Science 286, 971-4 (1999)).

[0092] The robustness of the assay employed was confirmed by calculating the Z' value for each plate of the library (Zhang et al. J Biomol Screen 4, 67-73 (1999); see FIG. IB for the average Z¹ value for the assay). [0093] Using this screening method, Z-5-(4-Hydroxyphenylidene)-2-imino-1,3- thiazolidin-4-one (referred to as "Mirin"; shown below) was identified as an inhibitor of DSB- induced ATM activation.

[0094] The IC₅₀ for inhibition of H2AX phosphorylation by Mirin was estimated to be 65 μM (FIG. ID).

[0095] Another compound (6-phenyl-2-thioxo-2,3-dihydro-4(1H)-pyrimidinone (compound IV), shown below) did not inhibit DSBs-induced ATM activation as seen by H2AX peptide phosphorylation.

[0096] To confirm the specificity of the assay for ATM kinase activity, ATM was immuno-depleted from extracts prior to addition of DSB-containing DNA and Mirin. In such ATM-depleted extracts, H2AX phosphorylation was significantly reduced. Moreover, the residual H2AX phosphorylation was insensitive to Mirin, demonstrating that the inhibitory effect of Mirin on H2AX phosphorylation was entirely ATM-dependent (FIG. ID). Mirin also inhibited ATM-dependent phosphorylation of downstream targets Nbsl and Chk2 29 (FIG. 2A) and the MRN-dependent autophosphorylation of ATM at amino acid position S 1981 in response to DSBs (FIG. 2B, lanes 3 and 5; FIG. 3C). These data establish that Xenopus cell- free extracts are valid and powerful system to identify small molecules that modulate the DNA damage response.

[0097] To identify the target of Mirin, extracts were depleted of the MRN complex using an antibody specific for MREIl in conjunction with two different amounts of biotin- labeled, DSB-containing DNA corresponding to 1.2xl0^πends/μl and 3.6xlθ^πends/μl, respectively (FIG. 2B). After removal of streptavidin-bound DNA, ATM activation was monitored in the resulting soluble fractions (FIG. 2B). H2AX peptide was phosphorylated to similar extents in mock-depleted extracts at both DNA concentrations (FIG. 2B, lanes 2 and 4). MRN depletion prevented ATM activation at 1.2xl0^πends/μl, but partial ATM activation was observed at 3.6xlθ^πends/μl (FIG. 2B, compare lanes 6 and 8). This confirms that MRN's requirement for ATM activation can be partially bypassed when the number of DNA breaks is increased. Notably, Mirin partially inhibited MRN-dependent ATM activation in mock- depleted extracts (FIG. 28, lanes 3 and 5). In contrast, Mirin had no effect on MRN- independent ATM activity triggered by 3.6xlO^π ends/μl (FIG. 28, lanes 7 and 9). This result indicates that Mirin inhibits MRN- dependent activation of ATM kinase but not ATM kinase activity.

[0098] This conclusion was confirmed by an in vitro assay with purified recombinant proteins and DNA. ATM was purified from mammalian cells as a catalytically inactive dimer (FIG. 2C; Lee et al., Science 308, 551-4 (2005)) or as a partly active monomer (FIG. 2D), as described by Lee et al. (Science 304, 93-6 (2004)). Activation of dimeric ATM by MRN and DNA was monitored using phosphorylation of recombinant p53 as a read-out with a phospho- specific antibody directed against Serl5 (Lee et al., Science 308, 551-4 (2005)). ATM activation was inhibited by Mirin with an IC50 of 15 μM (FIG. 2C). In contrast, the catalytic activity of monomeric ATM, which does not require MRN or DNA, was not inhibited by 50 μM Mirin (FIG. 2D). Taken together, these data establish unambiguously that Mirin inhibits MRN-dependent activation of ATM by specifically targeting the MRN complex.

[0099] Further studies have demonstrated that Mirin is a novel MREIl inhibitor. Moreover, data provided herein also indicate that the nuclease and DNA end- processing functions of MREl 1 are required for proper ATM activation.

[0100] The consequences of exposing mammalian cells to Mirin were also examined. Significant G2/M arrest was observed in exponentially growing TOSA4 and U2OS cells at Mirin concentrations of 50 μM and 100 μM. These data indicate that Mirin elicits a DNA damage checkpoint in G2. Further studies demonstrated that this G2/M arrest was ATM independent. Accordingly, compounds of the invention can arrest or inhibit growth of exponentially dividing cells at the G2 phase of the cell cycle.

[0101] Note however that when GI synchronized cells are exposed to γ-radiation from a ¹³⁷Cs source, Mirin abolished the 'irradiation-induced G2/M checkpoint and allowed cells to proceed through mitosis. These data illustrate that the compounds of the invention have two functions. First, Mirin can inhibit MRN and thereby stimulate DNA damage, which elicits a DNA damage checkpoint in the cell cycle, leading to reduced cell growth. Second, under some circumstances, Mirin can inhibit ATM signaling and thereby prevent a signaling cascade that gives rise to a G2/M checkpoint caused by irradiation. Nucleic Acid Inhibitors of MRE11-RAD50-NBS1 Expression

[0102] Nucleic acids that inhibit the expression of MREIl, RAD50, and/or NBSl can be used with the compositions and methods of the invention. Thus, in one embodiment, the compositions and methods of the invention include a nucleic acid that can inhibit the functioning of a MREI l, RAD50, and/or NBSl RNA. Nucleic acids that can inhibit the function of a MREIl, RAD50, and/or NBSl RNA can be generated from coding and non- coding regions of the MREl 1, RAD50, and/or NBSl genes. In the example provided herein, the MREIl -specific, RAD50-specific and/or NBSl-specific siRNA was based upon the coding sequence(s) available in public databases (for example the database provided by the National Center for Biotechnology Information, see website at ncbi.nlm.nih.gov).

[0103] For example, a sequence for human MREIl is available as NCBI accession number NM 005590 (gi: 56550106). This sequence is provided here as SEQ ID NO:3.

[0104] A sequence for human RAD50 is available as NCBI accession number NM 133482 (gi: 19924130). This sequence is provided here as SEQ ID NO:4.

[0105] A sequence for human NBSl is available as NCBI accession number BC146797 (gi: 148922297). This sequence is provided here as SEQ ID NO:5.

[0106] In some embodiments, the nucleic acid that can inhibit the function of a MREIl, RAD50, and/or NBSl RNA can be complementary to sequences near the 5' end of the MREIl, RAD50, and/or NBSl coding regions.

[0107] Hence, in some embodiments, the nucleic acid that can inhibit the functioning of a MREIl, RAD50 and/or NBSl RNA can be complementary to SEQ ID NO:3, 4, or 5. In other embodiments, nucleic acids that can inhibit the function of a MREIl, RAD50 and/or NBSl RNA can be complementary to the 5' ends of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or to variant MREIl, RAD50, and/or NBSl RNAs, or to MREIl. RAD50 and/or NBSl RNAs from other species (e.g., mouse, rat, cat, dog, goat, pig, or a monkey MREIl, RAD50 and/or NBSl RNAs).

[0108] A nucleic acid that can inhibit the functioning of a MREIl, RAD50 and/or NBSl RNA need not be 100% complementary to a selected region of SEQ ID NO:3, 4, or 5. Instead, some variability for example at least 80% complementarity, such as for example at least 90%, including 95%, 96%, 97%, 98% and 99% complementarity, in the sequence of the nucleic acid that can inhibit the functioning of a MREIl, RAD50 and/or NBSl RNA is permitted. For example, a nucleic acid that can inhibit the functioning of a human MREIl, RAD50, and/or NBSl RNA can be complementary to a nucleic acid encoding a mouse or rat MREIl, RAD50, and/or NBSl gene product. Nucleic acids encoding mouse MREIl, RAD50 and/or NBSl gene product, for example, can be found in the NCBI database.

[0109] Moreover, nucleic acids that can hybridize under moderately or highly stringent hybridization conditions are sufficiently complementary to inhibit the functioning of a MREIl, RAD50, and/or NBSl RNA and can be utilized in the compositions of the invention. Generally, stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (T,) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about I°C to about 2O°C lower than the thermal pointing point of the selected sequence, depending upon the desired degree of stringency as otherwise qualified herein. In some embodiments, the nucleic acids that can inhibit the functioning of MREIl, RAD50, and/or NBSl RNA can hybridize to a MREIl. RAD50 and/or NBSl RNA under physiological conditions, for example, physiological temperatures and salt concentrations.

[0110] Precise complementarity is therefore not required for successful duplex formation between a nucleic acid that can inhibit a MREIl, RAD50, and/or NBSl RNA and the complementary coding sequence of a MREIl, RAD50 and/or NBSl RNA. Inhibitory nucleic acid molecules that comprise, for example, 2, 3, 4, 5, or more stretches of contiguous nucleotides that are precisely complementary to a MREIl, RAD50 and/or NBSl coding sequence, each separated by a stretch of contiguous nucleotides that are not complementary to adjacent MREIl, RAD50 and/or NBSl coding sequences, can inhibit the function of MREIl, RAD50 and/or NBSl mRNA. In general, each stretch of contiguous nucleotides is at least 4, 5, 6, 7, 8, or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of a nucleic acid hybridized to a sense nucleic acid to estimate the degree of mismatching that will be tolerated between a particular nucleic acid for inhibiting expression of a particular MREl 1, RAD50 and/or NBSl RNA.

[0111] In some embodiments, a nucleic acid that can inhibit the function of an endogenous MREIl, RAD50 and/or NBSl RNA is an anti-sense oligonucleotide. The anti- sense oligonucleotide is complementary to at least a portion of the coding sequence of a gene comprising SEQ ID NO:3, 4, or 5. Such anti- sense oligonucleotides are generally at least six nucleotides in length, but can be about 8, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Longer oligonucleotides can also be used. MREIl, RAD50, and/or NBSl anti-sense oligonucleotides can be provided in a DNA construct, or expression cassette and introduced into cells whose division is to be decreased, for example, into cells expressing MREIl, RAD50, and/or NBSl, including mast cells.

[0112] In one embodiment of the invention, expression of a MREIl, RAD50, and/or NBSl gene is decreased using a ribozyme. A ribozyme is an RNA molecule with catalytic activity. See, e.g., Cech, 1987, Science 236: 1532-1539; Cech, 1990, Ann. Rev. Biochem. 59:543-568; Cech, 1992, Curr. Opin. Struct. Biol. 2: 605-609; Couture and Stinchcomb, 1996, Trends Genet. 12: 510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (see, e.g., Haseloff et al., U.S. Pat. No. 5,641,673).

[0113] Nucleic acids complementary to SEQ ID NO:3, 4, or 5 can be used to generate ribozymes that will specifically bind to mRNA transcribed from a MREIl, RAD50 and/or NBSl gene. Methods of designing and constructing ribozymes that can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988), Nature 334:585-591). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201). The target sequence can be a segment of about 10, 12, 15, 20, or 50 contiguous nucleotides selected from a nucleotide sequence having SEQ ID NO: 3, 4, or 5. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related; thus, upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

[0114] RNA interference (RNAi) involves post-transcriptional gene silencing (PTGS) induced by the direct introduction of dsRNA. Small interfering RNAs (siRNAs) are generally 21-23 nucleotide dsRNAs that mediate post-transcriptional gene silencing. Introduction of siRNAs can induce post-transcriptional gene silencing in mammalian cells, siRNAs can also be produced in vivo by cleavage of dsRNA introduced directly or via a transgene or virus. Amplification by an RNA-dependent RNA polymerase may occur in some organisms. siRNAs are incorporated into the RNA-induced silencing complex, guiding the complex to the homologous endogenous mRNA where the complex cleaves the transcript.

[0115] Rules for designing siRNAs are available. See, e.g., Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). Duplexes of 21 -nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature All: 494- 498; J. Harborth, S. M. Elbashir, K. Vandenburgh, H. Manninga, S. A. Scaringe, K. Weber and T. Tuschl (2003). Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing, Antisense Nucleic Acid Drug Dev. 13: 83-106.

[0116] Thus, an effective siRNA can be made by selecting target sites within SEQ ID NO:3, 4, or 5 that begin with AA, that have 3' UU overhangs for both the sense and antisense siRNA strands, and that have an approximate 50% G/C content.

[0117] For example, a siRNA of the invention that can hybridize to MREIl, RAD50 and/or NBSl nucleic acids can be homologous or complementary to one of the following sequences,

MREIl:

ACAGGAGAAGAGATCAACT (SEQ ID NO:6) (see, Chai et al. EMBO REP 7(2): 225-230, (2006));

CCTGCCTCGAGTTATTAAG (SEQ ID NO:7) (Takemura et al. J Biol Chem 281(41): 30814-30823 (2006));

CTGCGAGTGGACTATAGTG (SEQ ID NO:8 ) (Takemura et al. / Biol Chem 281(41):30814-30823 (2006)));

GATGCCATTGAGGAATTAG (SEQ ID NO:9) (Takemura et al. J Biol Chem 281(41): 30814-30823 (2006));

GAGCAUAACUCCAUAAGUANTNT (SEQ ID NO: 10) (Adams et al. Oncogene 25(28): 3894-3904 (2006));

GCUAAUGACUCUGAUGAUA (SEQ ID NO: 11) (Myers & Cortez, / Biol Chem 281(14): 9346-9350 (2006))

RAD50:

GGAGAAGGAAATACCAGAA (SEQ ID NO: 12) (see, Chai et al. EMBO REP 7(2): 225-230, (2006));

GCAGACTTAGACAGGACCC (SEQ ID NO:13) (Zhong et al. Hum MoI Genet 14(18): 2685-2693 (2005));

GCUCAGAGAUUGUGAAAUG (SEQ ID NO: 14) (Myers & Cortez, / Biol Chem 281(14): 9346-9350 (2006))

NBSl:

CAGGAGGAAGATGTCAATG (SEQ ID NO: 15) (see, Chai et al. EMBO REP 7(2): 225-230, (2006));

GAAGAAACGUGAACUCAAG (SEQ ID NO: 16) (Myers & Cortez, / Biol Chem 281(14): 9346-9350 (2006)) GGCGUGUCAGUUGAUGAAANTT (SEQ ID NO: 17 ) (see, Zhang et al. Cancer Res 65(13): 5544-5553 (2005)).

[0118] These nucleic acids can be used in the compositions and methods of the invention either alone or in combination with the compounds described herein. Treatment Methods

[0119] According to the invention, inhibitors of MREIl, RAD50 and/or NBSl are useful for reducing cancer cell and tumor growth, which improves of the survival of mammals with cancer and/or tumors. Thus, the invention provides methods of treating cancer in a mammal that involve administering to the mammal a therapeutically effective amount of a compound or nucleic acid that can inhibit MEREIl, RAD50 and/or NBSl activity or expression.

[0120] According to the invention, a variety of cancers can be treated or prevented including, but not limited to: carcinomas such as breast, bladder, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoxanthoma, thyroid follicular cancer and Kaposi's sarcoma.

[0121] In some embodiments, the cancer cells have a defect in a DNA repair pathway or a DNA damage response mechanism. Many cancers harbor signs of genomic instability. Such instability stems from different causes including defects in the DNA damage response: DNA repair (BRCA 1/2, FA, NBSl), cell cycle checkpoints (p53, ATM, Chk2) or apoptosis (p53) and/or increase in DNA damage following replication stress (Myc) (Kastan et al., Nature 432: 316 (Nov 18, 2004); D. Domingues-Sola et al., Nature, In press (2007)). Such cancer cells show higher steady state levels of DNA damage than normal cells (Bartkova et al., Nature 434, 864 (Apr 14, 2005)), which puts tumor cells at risk for acquiring lethal mutations. These cells therefore rely more heavily than normal cells on DNA repair mechanisms for survival. Thus, repair of DNA double- strand breaks (DSBs) is critical for the viability of cancer cells with such defects DNA repair pathways or DNA damage response mechanisms.

[0122] Because these cancer cells are so dependent upon remaining DNA repair mechanisms they are even more susceptible to the present compositions and methods, which inhibit the DNA damage response. Thus, mammals with cancer or tumor cells that have one or more defects in DNA repair pathways or DNA damage response mechanisms are beneficially treated with the present compositions and methods.

[0123] Double stranded breaks in DNA are repaired via two major pathways in mammalian cells: non-homologous end joining (NHEJ) and homology-dependent DNA repair (HDR). Homologous repair operates during S-phase or G2 when a homologous sequence of the damaged site is available. Homology-dependent repair at a DNA double-strand break starts with the localization of the MRE11-RAD50-NBS1 (MRN) complex to the double stranded break. The break is resected, long-range chromatin modifications take place and the resected DNA invades the homologous sequence in a Rad51/52 dependent reaction. Additional factors are then required, including Rad paralogs and BRCAl/2 in vertebrates.

[0124] Cancer cell "addiction to repair" was recently validated for cells carrying mutations in BRCAI or BRCAZ that are defective in double-stranded break repair of DNA by homology-dependent DNA repair. In particular, BRCA^-7- cells become dependent on otherwise non-essential DNA repair pathways for cell survival as seen by their exquisite sensitivity to poly(ADP)ribose polymerase (PARP) inhibitors (Bryant et al., Nature 434: 913 (Apr 14, 2005); Farmer et al., Nature 434: 917 (Apr 14, 2005)). The sensitivity is thought to be due to a synergy between the single-strand (PARP inhibition) and homology-dependent DNA repair (BRCAl , BRC A2) repair defects.

[0125] Thus, according to the invention, the intrinsic genomic instability of many types of cancer cells makes them more reliant on DNA repair by homology-dependent DNA repair than normal cells. Cancer cells harboring defects in the maintenance of genome stability therefore become more sensitive to inhibition of homology-dependent DNA repair alone. Moreover, inhibiting homology-dependent DNA repair in cancer cells will make them more dependent on other repair pathways and, as a consequence, more sensitive to irradiation or PARP inhibition. Therefore, cancer and tumor cells with defects in a DNA repair pathway or a DNA damage response mechanism can readily be treated with the present compositions and methods. [0126] Examples of proteins involved in the maintenance of genome stability following double-stranded breaks in DNA that are associated with cancer predisposition and defects in neural development are shown in Table 1 below.

Table 1: Examples of Proteins Involved in Genome Stability

.

[0127] According to the invention, the present compositions and methods are useful for treating cancers correlated with mutations in the genes encoding such proteins.

[0128] Moreover, as illustrated herein, inhibition of MREIl, and/or other components of the MRN complex, arrests exponentially growing cells in the G2/M phase of the cell cycle. Thus, mammals with exponentially growing tumor or cancer cells can also be beneficially treated with the present compositions and methods even when the cancer cells are not known to have one or more defects in DNA repair pathways or DNA damage response mechanisms. [0129] The compounds and nucleic acids of the invention can also be administered with other chemotherapeutic agents. For example, the compounds and nucleic acids of the invention can also be administered with an anti-microtubule agent, platinum coordination complex, alkylating agent, antibiotic, topoisomerase II inhibitor, antimetabolite, topoisomerase I inhibitor, hormone, signal transduction pathway inhibitor, non-receptor tyrosine kinase angiogenesis inhibitor, immunotherapeutic agent, proapoptotic agent, cell cycle signaling inhibitor or a combination thereof.

[0130] Other chemotherapeutic agents that can be administered with the compounds and/or nucleic acids of the invention include altretamine, aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, calcium folinate, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, crisantaspase, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, vinorelbine, tamoxifen, 4-(3-chloro-4- fluorophenylamino)-7-methoxy-6-(3-(4-α-morpholinyl)propoxy)quinazoline, 4-(3- ethynylphenylamino)-6,7-bis(2-methoxyethoxy)quinazoline, hormones, steroids, 17α- ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, Zoladex, antiangiogenics, matrix metalloproteinase inhibitors, VEGF inhibitors, ZD6474, SU6668, SUl 1248, anti-Her-2 antibody (e.g., ZD1839 and OS1774), EGFR inhibitor, EKB-569, Imclone antibody C225, src inhibitor, bicalutamide, epidermal growth factor inhibitors, Her-2 inhibitor, MEK-I kinase inhibitor, MAPK kinase inhibitor, Pl 3 inhibitor, PDGF inhibitor, combretastatin, MET kinase inhibitor, MAP kinase inhibitors, inhibitor of non-receptor and receptor tyrosine kinases (imatinib), inhibitor of integrin signaling, inhibitor of insulin-like growth factor receptors, an EGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin, carboplatin, cimetidine, carminomycin, mechlorethamine hydrochloride, pentamethylmelamine, thiotepa, teniposide, cyclophosphamide, chlorambucil, demethoxyhypocrellin A, melphalan, ifosfamide, trofosfamide, Treosulfan, podophyllotoxin, etoposide phosphate, teniposide, etoposide, leurosidine, leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, Chloroambucil, megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU), lovastatin, l-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin, thiotepa, phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin, cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine, doxorubicin hydrochloride, leucovorin, mycophenoloc acid, daunorubicin, deferoxamine, floxuridine, doxifluridine, ratitrexed, idarubicin, epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate, mitomycin C, actinomycin D, safracins, saframycins, quinocarcins, discodermolides, vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel, tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR, estramustine, estramustine phosphate sodium, flutamide, bicalutamide, buserelin, leuprolide, pteridines, diyneses, levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride, betamethosone, gemcitabine hydrochloride, verapamil, VP- 16, altretamine, thapsigargin, topotecan and combinations thereof. Screening Methods

[0131] Screening methods can be used to identify agents useful for treating cancer and for monitoring or assessing the efficacy and dosages of agents already identified as anti-cancer agents.

[0132] One assay to specifically monitor the rapid activation of ATM by double- stranded breaks in DNA is provided as follows. Cytosol or membrane-free cytosol extracts from Xenopus eggs are employed and can be prepared as described in Costanzo et al., Methods in Molec. Biol. 280: 213-227 (2004). These extracts are supplemented with DNA having double- stranded breaks. Different sources of DNA and various sizes of DNA can be employed. For example, such DNA with double- stranded breaks can be generated by plasmid or λ DNA digested with restriction enzymes, or by PCR amplification. Double stranded DNA fragments of about 50 base pairs to about 2000 base pairs, or about 150 base pairs to about 1,000 base pairs have been used successfully. Note that double stranded DNA fragments larger than 200 base pairs very rapidly assemble into nucleosomes (Almouzni & Wolffe, Exp Cell Res 205(1): 1-15 (1993)). After exposure to DNA with double-stranded breaks, an aliquot of the reaction is then diluted 10-fold into kinase buffer containing ³²P-γATP and a reporter peptide derived from the C-terminal tail of histone H2AX (AVGKKASQASQEY, SEG ID NO:2). The reaction is spotted on phosphocellulose paper and washed before counting (due to its Lys-Lys sequence, this peptide has very high affinity for phosphocellulose).

[0133] ATM phosphorylation specificity was established by depleting extracts with three different ATM polyclonal antibodies or with an immobilized peptide derived from the C- terminal 22 amino acid ATM-binding domain of Xenopus NBSl (Falck et al. Nature 434(7033): 605-611 (2005)). Both methods of ATM depletion result in the inhibition of H2AX peptide phosphorylation (see FIG. 6A-B).

[0134] This assay has been adapted to screen libraries of small molecules for inhibitors of the MRN-ATM pathway using high-throughput screening methods. For example, a 96-well plate format can be used instead of performing the kinase assay in a tube. When using such a microtiter plate, the assays are performed in small volume (2 μl) in V-shaped 96-well plates. After the phosphorylation reaction has occurred, the assay mixtures are transferred to P81 phosphocellulose coated 96-well plates. These plates are then rinsed and the bound radiolabeled H2AX (SEQ ID NO:2) peptide is detected with a Phosphorlmager. An example of a plate is shown in FIG. IA and FIG. 8.

[0135] Thus, the invention is also directed to screens to identify inhibitors and agonists of the MRN-ATM pathway. Such screens are performed by using low concentration of DNA with double- stranded breaks (e.g., 2 ng/μl for screening for agonists) in the phosphorylation assay described above. Such low concentrations of damaged DNA only partially activates ATM in extracts containing MRN (FIG. 8).

[0136] This assay and modifications thereof have been used to monitor ATM activation. In particular, studies have shown that ATM activation by DNA with double-stranded breaks occurs in two steps (Dupre et al. Nat Struct MoI Biol 13(5): 451-457 (2006). In the first step, dimeric ATM is recruited to damaged DNA and dissociates into monomers. The MRN complex facilitates this process by tethering DNA, thereby increasing the local concentration of damaged DNA. Significantly, increasing the concentration of damaged DNA can bypass the requirement for the DNA-tethering activity of MRN. This permits generation of ATM molecules in a monomeric state, primed for activation. ATM monomers generated in the absence of MRN are not phosphorylated on Serine 1981 and are only partially active (FIG. 7A- B).

[0137] In a second step, the ATM-binding domain of Nbsl is required and sufficient to convert unphosphorylated ATM monomers into fully enzymatically active monomers in the absence of DNA (FIG. 7 A-B). These studies helped to clarify the mechanism of ATM activation in normal cells and explains why cells from patients with Ataxia Telangiectasia-like disorder and Nijmegen Breakage Syndrome display partial ATM activation at high doses of ionizing radiation. This second step also establishes that the interaction between ATM and the C-terminal 22 amino acids of the NBSl protein is triggering an activating event, possibly a critical conformational change, within the ATM monomers.

[0138] Accordingly, the invention provides a method of identifying an agent that can modulate the MRN-ATM pathway. Such a method involves contacting a assay mixture with a test agent, determining whether phosphorylation of an H2AX (SEQ ID NO:2) peptide increases or decreases relative to a control assay that does not contain the test agent, and identifying an agent that modulates the MRN-ATM pathway when that agent increases or decreases the phosphorylation of the H2AX (SEQ ID NO:2) peptide relative to the control assay. The assay mixture contains the H2AX (SEQ ID NO:2) peptide, a Xenopus oocyte cytoplasmic extract, ATP where the terminal phosphate has a detectable label and DNA with double-stranded breaks. In some embodiments, for example, a 96-well plate with V-shaped wells can be used where the assays are performed in small volume (about 1-5 microliters, or about 1-3 microliters or about 2 microliters). After the phosphorylation reaction has occurred, the assay mixtures are transferred to P81 phosphocellulose coated 96-well plates. These plates are then rinsed and the bound labeled H2AX (SEQ ID NO:2) peptide is detected using a device that permits detection of the labeled phosphate incorporated onto the labeled H2AX (SEQ ID NO:2) peptide (e.g. a Phosphorlmager when γ-³²P-ATP is used). Compositions

[0139] The compounds and/or nucleic acids of the invention are administered to sensitize cancer cells to chemotherapy and/or to improve the efficiency of treatment and achieve a reduction in at least one symptom associated with a cancerous condition or other disease associated with inappropriate cellular growth. In some embodiments, the compounds and/or nucleic acids are administered to reduce cancer or tumor cell growth or to sensitize cells such that other agents which are anti-tumor agents or chemotherapeutic agents, or the like can work effectively. Other agents such as anti-tumor agents, chemotherapeutic agents, analgesics, etc. can be administered with the compounds and nucleic acids of the invention or be included in the compositions or co-administered in accordance with the methods described herein.

[0140] To achieve the desired effect(s), the compounds, nucleic acids, and combinations with other agents (e.g., chemotherapeutic agents), may be administered as single or divided dosages. For example, the compounds and nucleic acids can be administered in dosages of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg, or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, including 2 mg/kg to about 10 mg/kg, although other dosages may provide beneficial results. In some embodiments, the compounds of the invention are administered so as to achieve blood concentration levels of about 0.1 μM to about 100 μM, including 0.1 to 1 μM, or 1 to 10 μM. In other embodiments, the compounds of the invention are administered so as to achieve blood concentration levels of about 1 μM to about 100 μM, including 10 μM to 50 μM.

[0141] The amount administered will vary depending on various factors including, but not limited to, the compound or nucleic acid chosen, the disease, the weight, the physical condition, the health, the age of the mammal, whether prevention or treatment is to be achieved, if the compound or nucleic acid is administered with a chemotherapeutic agent, and which chemotherapeutic agent is co-administered with the compound or nucleic acid. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.

[0142] Administration of the therapeutic agents in accordance with the present invention may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of compounds and nucleic acids of the invention optionally with other anticancer agents may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.

[0143] To prepare the composition, compounds, nucleic acids and agents are synthesized or otherwise obtained, purified as necessary or desired and then lyophilized and stabilized. The compound, or nucleic acid can then be adjusted to the appropriate concentration, and optionally combined with other agents (e.g. chemotherapeutic agents). The absolute weight of a given compound, or nucleic acid included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one compound or nucleic acid of the invention, or a plurality of compounds and nucleic acids can be administered. Alternatively, the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.

[0144] Daily doses of the compounds or nucleic acids of the invention can vary as well. Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.

[0145] Thus, one or more suitable unit dosage forms comprising the therapeutic compounds or nucleic acids of the invention can be administered by a variety of routes including oral, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. In some embodiments, the compounds or nucleic acids are administered locally to tumor or cancer sites.

[0146] The therapeutic agents may also be formulated for sustained release (for example, using microencapsulation, see WO 94/07529, and U.S. Patent No. 4,962,091). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agents with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[0147] When the therapeutic agents of the invention are prepared for oral administration, they are generally combined with a pharmaceutically acceptable carrier, diluent, or excipient to form a pharmaceutical formulation, or unit dosage farm. For oral administration, the therapeutic agents may be present as a powder, a granular formulation, a solution, a suspension, an emulsion, or in a natural or synthetic polymer or resin for ingestion of the active ingredients from a chewing gum. The therapeutic agents may also be presented as a bolus, electuary or paste. Orally administered therapeutic agents of the invention can also be formulated for sustained release, e.g. the therapeutic agents can be coated, micro-encapsulated, or otherwise placed within a sustained delivery device. The total active ingredients in such formulations comprise from 0.001 to 99.9% by weight of the formulation.

[0148] By "pharmaceutically acceptable" is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

[0149] Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well-known and readily available ingredients. For example, the therapeutic agents can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, solutions, suspensions, powders, aerosols and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include buffers, as well as fillers and extenders such as starch, cellulose, sugars, mannitol, and silicic derivatives. Binding agents can also be included such as carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropyl methylcellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone. Moisturizing agents can be included such as glycerol, disintegrating agents such as calcium carbonate and sodium bicarbonate. Agents for retarding dissolution can also be included such as paraffin. Resorption accelerators such as quaternary ammonium compounds can also be included. Surface-active agents such as cetyl alcohol and glycerol monostearate can be included. Adsorptive carriers such as kaolin and bentonite can be added. Lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols can also be included. Preservatives may also be added. The compositions of the invention can also contain thickening agents such as cellulose and/or cellulose derivatives. They may also contain gums such as xanthan, guar, or carbo gum, or gum arabic, or alternatively polyethylene glycols, bentones and montmorillonites, and the like.

[0150] For example, tablets or caplets containing the therapeutic agents of the invention can include buffering agents such as calcium carbonate, magnesium oxide and magnesium carbonate. Caplets and tablets can also include inactive ingredients such as cellulose, pregelatinized starch, silicon dioxide, hydroxy propyl methyl cellulose, magnesium stearate, microcrystalline cellulose, starch, talc, titanium dioxide, benzoic acid, citric acid, corn starch, mineral oil, polypropylene glycol, sodium phosphate, zinc stearate, and the like. Hard or soft gelatin capsules containing at least one therapeutic agent of the invention can contain inactive ingredients such as gelatin, microcrystalline cellulose, sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like, as well as liquid vehicles such as polyethylene glycols (PEGs) and vegetable oil. Moreover, enteric-coated caplets or tablets containing one or more therapeutic agents of the invention are designed to resist disintegration in the stomach and dissolve in the more neutral to alkaline environment of the duodenum.

[0151] The therapeutic agents of the invention can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous, intraperitoneal or intravenous routes. The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension or salve.

[0152] Thus, the therapeutic agents may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion containers or in multi- dose containers. As noted above, preservatives can be added to help maintain the shelve life of the dosage form. The active compounds, nucleic acids and other ingredients may form suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active compounds, nucleic acids and other ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0153] These formulations can contain pharmaceutically acceptable carriers, vehicles and adjuvants that are well known in the art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint, chosen, in addition to water, from solvents such as acetone, ethanol, isopropyl alcohol, glycol ethers such as the products sold under the name "Dowanol," polyglycols and polyethylene glycols, C₁-C₄ alkyl esters of short-chain acids, ethyl or isopropyl lactate, fatty acid triglycerides such as the products marketed under the name "Miglyol," isopropyl myristate, animal, mineral and vegetable oils and polysiloxanes.

[0154] It is possible to add, if desired, additional ingredients chosen from chemotherapeutic agents, antioxidants, analgesics, surfactants, other preservatives, film- forming, keratolytic, or comedolytic agents, perfumes, flavorings and colorings. Antioxidants such as t-butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene and a- tocopherol and its derivatives can be added.

[0155] Additionally, the compounds and nucleic acids are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the therapeutic agents, for example, in a particular part of the intestinal or respiratory tract, possibly over a period of time. Coatings, envelopes, and protective matrices may be made, for example, from polymeric substances, such as polylactide-glycolates, liposomes, microemulsions, microparticles, nanoparticles, or waxes. These coatings, envelopes, and protective matrices are useful to coat indwelling devices, e.g., stents, catheters, peritoneal dialysis tubing, draining devices and the like.

[0156] For topical administration, the therapeutic agents may be formulated as is known in the art for direct application to a target area. Forms chiefly conditioned for topical application take the form, for example, of creams, milks, gels, dispersion, or microemulsions, lotions thickened to a greater or lesser extent, impregnated pads, ointments, or sticks, aerosol formulations (e.g., sprays or foams), soaps, detergents, lotions, or cakes of soap. Other conventional forms for this purpose include wound dressings, coated bandages, or other polymer coverings, ointments, creams, lotions, pastes, jellies, sprays, and aerosols. Thus, the therapeutic agents of the invention can be delivered via patches, or bandages for dermal administration. Alternatively, the compounds and/or nucleic acids can be formulated to be part of an adhesive polymer, such as polyacrylate or acrylate/vinyl acetate copolymer. For long- term applications, it might be desirable to use microporous and/or breathable backing laminates, so hydration or maceration of the skin can be minimized. The backing layer can be any appropriate thickness that will provide the desired protective and support functions. A suitable thickness will generally be from about 10 to about 200 microns.

[0157] Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. The therapeutic agents can also be delivered via iontophoresis, e.g., as disclosed in U.S. Patent Nos. 4,140,122; 4,383,529; or 4,051,842. The percent by weight of a therapeutic agent of the invention present in a topical formulation will depend on various factors, but generally will be from 0.001% to 95% of the total weight of the formulation, and typically 0.01-85% by weight.

[0158] Drops, such as eye drops or nose drops, may be formulated with one or more of the therapeutic agents in an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs. Drops can be delivered via a simple eye dropper-capped bottle, or via a plastic bottle adapted to deliver liquid contents dropwise, via a specially shaped closure.

[0159] The therapeutic agents may further be formulated for topical administration in the mouth or throat. For example, the active Ingredients may be formulated as a lozenge further comprising a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the composition of the present invention in a suitable liquid carrier.

[0160] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0. [0161] The therapeutic agents of the invention can also be administered to the respiratory tract. Thus, the present invention also provides aerosol pharmaceutical formulations and dosage forms for use in the methods of the invention. In general, such dosage forms comprise an amount of at least one of the agents of the invention effective to treat or prevent the clinical symptoms of a specific cancer, tumor, indication or related disease. Any statistically significant attenuation of one or more symptoms of a cancer that has been treated pursuant to the method of the present invention is considered to be a treatment of such cancer within the scope of the invention.

[0162] Alternatively, for administration by inhalation or insufflation, the composition may take the form of a dry powder, for example, a powder mix of the therapeutic agents and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges, or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator, insufflator, or a metered- dose inhaler (see, for example, the pressurized metered dose inhaler (MDI) and the dry powder inhaler disclosed in Newman, S. P. in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds., pp. 197-224, Butterworths, London, England, 1984).

[0163] Therapeutic agents of the present invention can also be administered in an aqueous solution when administered in an aerosol or inhaled form. Thus, other aerosol pharmaceutical formulations may comprise, for example, a physiologically acceptable buffered saline solution containing between about 0.1 mg/ml and about 100 mg/ml of one or more of the therapeutic agents of the present invention specific for the indication or disease to be treated. Dry aerosol in the form of finely divided solid compound or nucleic acid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention. Compounds or nucleic acids of the present invention may be formulated as dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 μM, alternatively between 2 and 3 μM. Finely divided particles may be prepared by pulverization and screen filtration using techniques well known in the art. The particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a powder. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular infection, indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations. [0164] For administration to the upper (nasal) or lower respiratory tract by inhalation, the therapeutic agents of the invention are conveniently delivered from a nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Nebulizers include, but are not limited to, those described in U.S. Patent Nos. 4,624,251; 3,703,173; 3,561,444; and 4,635,627. Aerosol delivery systems of the type disclosed herein are available from numerous commercial sources including Fisons Corporation (Bedford, Mass.), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co., (Valencia, CA). For intranasal administration, the therapeutic agent may also be administered via nose drops, a liquid spray, such as via a plastic bottle atomizer or metered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Medihaler (Riker).

[0165] Furthermore, the active ingredients may also be used in combination with other therapeutic agents, for example, chemotherapeutic agents, pain relievers, anti- inflammatory agents, antihistamines, antimicrobial agents, bronchodilators and the like, whether for the conditions described or some other condition.

[0166] The present invention further pertains to a packaged pharmaceutical composition for sensitizing cancer cells to chemotherapeutic agents such as a kit or other container. The kit or container holds a therapeutically effective amount of a pharmaceutical composition for sensitizing cancer cells to chemotherapeutic agents, and instructions for using the pharmaceutical composition for sensitizing cancer cells to chemotherapeutic agents. The pharmaceutical composition includes at least one compound or nucleic acid of the present invention, in a therapeutically effective amount such that cancer cells are sensitized to chemotherapeutic agents. The composition can also contain one or more anti-tumor agents or a chemotherapeutic agents.

[0167] In another embodiment, the invention provides a packaged pharmaceutical composition for inhibiting or killing cancer cells and/or sensitizing cancer cells to chemotherapeutic agents. The kit or container holds a therapeutically effective amount of a pharmaceutical composition of the invention and instructions for using the pharmaceutical composition for inhibiting or killing cancer cells and/or sensitizing cancer cells to chemotherapeutic agents. The pharmaceutical composition includes at least one compound or nucleic acid of the present invention, in a therapeutically effective amount such that a cancer cell is inhibited, killed or sensitized to chemotherapy. [0168] The invention will be further described by reference to the following detailed examples, which are given for illustration of the invention, and are not intended to be limiting thereof.

Example 1: Materials and Methods

[0169] This Example describes certain materials and methods used in the development of the invention.

Chemicals

[0170] Caffeine was purchased from Sigma (C-0750; St Louis, MO) and dissolved in 10 mM PIPES at pH 8.0. αP³²-ddATP, α³²P-dCTP, and γ³²P-ATP (10 μCi/μl) were purchased from Amersham Biosciences (Piscataway, NJ) and MD single- stranded DNA was from New England Biolabs (Ipswich, MA).

Xenopus extracts

[0171] Membrane-free egg cytosols (HSS) were prepared in the presence of cycloheximide as described by Smythe et al. (Methods Cell Biol 35, 449-68 (1991)). AU incubations were performed at 22°C.

Preparation of U2OS cell lysates

[0172] U2OS cells were synchronized in Gl/S using double Thymidine block (see below). Thirty minutes after addition of Mirin, cells were mock-treated or irradiated (10 Gy) from a Cesium-137 source in presence of 2 mM Thymidine to maintain cells at Gl/S border. Thirty minutes later, cells were harvested and washed twice with cold IX PBS. Cell pellets were resuspended in lysis buffer (50 mM Tris, pH 7.6, 100 mM NaCl, 1% SDS, 5 mM DTT, Ix protease inhibitor cocktail (Sigma). Lysates were heated 5 minutes at 95°C, protein quantified by Bradford Assay and the equivalent of 30 μg protein was loaded onto 3-8% NuPage Tris- Acetate gel (Invitrogen), followed by Western Blotting. The chemiluminescent signal was quantified using densitometer and ImageQuant software.

Immunodepletions and Antibodies

[0173] For Xenopus experiments, the MREIl, NBSl and ATM antibodies employed were as described by Dupre et al. (Nat Struct MoI Biol 13, 451-7 (2006)). CHK2 antibody was a kind gift of Dr. H. Lindsay (U. of Lancaster). For immunodepletions, extracts were incubated three times with protein A-Sepharose beads (GE Healthcare) coupled to anti-XMREll serum (ratio of extract/beads/serum = 1:1:1) or preimmune serum previously diluted in PBS (Mock depletion). [0174] For mammalian cells, antibodies directed against the phosphorylated form of ATM and total ATM were purchased from Rockland Immunochemicals, Inc. HRP- conjugated secondary antibodies were from Jackson ImmunoResearch laboratories.

Small molecule library screen

[0175] Small molecule library: The small molecule library was obtained from Chembridge Corporation (San Diego, CA). This 10,000 compound "Diverset" library is available in a 96-well format, with 80 compounds/plate. The library was re -plated and the compounds diluted to achieve 100 μM concentration for screening. High compound concentrations were used for the screen because of the high lipid and protein content of Xenopus extracts resulting in the non-specific sequestration of the small molecules.

[0176] Screening procedure: Extracts were incubated with 5 ng/μl of DSB- containing DNA (Haelll-digested pBlueScript DNA) for 20 minutes at 22°C. 2 μl aliquots of extracts were transferred to 96-well V-bottom plates containing the small molecule library on ice. 20 μl of kinase buffer was added to each well (2 μl 1 mM ATP, 1 μl of H2AX or control peptide 10 mg/ml, 0.4μl γ³²P-ATP at 10 μCi/μl, 16.6 μl EB: 80 mM β-glycero-phosphate, 10 mM MgCI₂, 20 mM HEPES, 1 mM DTT). The samples were mixed thoroughly and incubated for 10 minutes at 30 C.

[0177] Processing of 96-well P81 plates (Millipore) was performed on Flusher plate washer (Flushtec, Cathedral City, CA). The P81 phosphocellulose 96-well plates were pre- washed with 100/μl of wash buffer containing 0.75% phosphoric acid. The kinase reactions were stopped by placing the plates on ice and adding 20 μl of 0.75% phosphoric acid. The reactions were then transferred to the P81 plates. Binding was allowed for 30 seconds and the vacuum was applied. The plates were then washed 5 times in 300 μl of 0.75% phosphoric acid and dried with vacuum. The P81 plate backing was removed, excess liquid absorbed, and the P81 paper was exposed to a phosphor storage screen for 24 hrs, then processed for image analysis with a Phosphorlmager (Molecular Dynamics, GE Healthcare). The phosphorylation of H2AX peptide was calculated for each sample according to the formula (value of sample - average of value of negative controls with control peptide) / (average value of 80 samples- average value of negative control).

[0178] Percentages of inhibition of H2AX peptide phosphorylation were calculated according to the formula: (1 -((average of sample value -average of value of negative control) / (average value of positive control -average value of negative control))* 100).

[0179] In all the other experiments monitoring ATM activation in extracts, ATM was activated by incubating extracts with biotinylated DNA that was bound to streptavidin beads as described in Dupre et al. (Nat Struct MoI Biol 13, 451-7 (2006)). The reaction was spotted on P81 phosphocellulose filter papers (Upstate Biotechnology) and the radioactivity incorporated was quantified by scintillation. The number of cpm incorporated into control peptide was then subtracted from the number of cpm incorporated into the H2AX peptide, and the values were normalized to untreated extracts.

Biotinylated DNA pull-down experiments

[0180] Non-biotinylated and biotinylated 150 bp DNA fragments were generated by PCR using M 13 single stranded DNA template and Pfu polymerase as previously described in Dupre et al. (Nat Struct MoI Biol 13, 451-7 (2006)). Non-biotinylated radioactive DNA was also produced by PCR in the presence of α-P -dCTP. Unincorporated nucleotides were removed using Qiagen purification kit. Biotinylated DNA was coupled to streptavidin-coated magnetic beads (M-280, Dynal Biotech) as described in Dupre et al. (Nat Struct MoI Biol 13,451-7 (2006)). Beads were washed six times with ELB buffer (10 mM HEPES, pH 7.7; 2.5 mM MgCl₂; 0.05 mM KCl and 250 mM sucrose) and then incubated with extracts for 10 minutes at a final concentration of 1.2xlO^π or 3.6xlO^π ends/μl. DNA and extracts were then separated according to the manufacturer's instructions. DNA was washed six times with ELB supplemented with 0.1% (v/v) Triton X-100. Proteins were analyzed by Western blot in both soluble fractions and DNA fractions using NuPage 3-8% Tris-acetate SDS polyacrylamide gels (Invitrogen).

[0181] For extract fractionation, 50 μl of extracts were loaded on a Superose 6 gel- filtration column and 1 ml fractions were collected. 200 μl of each fraction was then precipitated with trichloroacetic acid and analyzed on a 6% SDS-PAGE gel by Western blot using an antibody directed against MREIl.

[0182] For FLAG pull down experiments, 5 μl of WT FLAG-MRN was added for 15 minutes to extracts, in the presence DMSO or Mirin. Recombinant complexes were then isolated with 5 μl of M2-FLAG resin. Resulting supernatants (1 μl) and beads (5 μl) were loaded on a 8% SDS page and immunoblotted using an antibody against FLAG (Sigma), MREIl (Dupre et al. (2006)) or RAD50 (Santa-Cruz).

[0183] For DNA tethering assays, streptavidin-bound biotinylated DNA and non- biotinylated radioactive DNA were incubated in extracts for 30 minutes. Biotinylated DNA was then pulled down, washed 6 times with ELB buffer supplemented with 0.1%(v/v) triton XlOO, and the radioactivity associated with biotinylated DNA counted by scintillation.

Production and purification of recombinant FLAG MRN complex [0184] Human FLAG-MRN complex was expressed from pTP (MREIl), pTP (RAD50), and pTP (FLAG-NBSl) baculovirus constructs in SF9 insect cells as described by Lee & Paull (Methods Enzymol 408, 529-39 (2006)). Proteins were eluted from M2- FLAG agarose resin (Sigma) with 20 mM Tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.1 % Triton X-100, 0.1% IGEPAL supplemented with 0.1mg/ml FLAG peptide (Sigma) and dialysed against ELB buffer.

ATM protein kinase assays with recombinant proteins

[0185] Monomeric ATM, dimeric ATM and MRN were purified as described by Lee & Paull (Methods Enzymol 408, 529-39 (2006)). Monomeric and dimeric ATM activity was assayed in vitro as described by Lee & Paull (Science 304, 93-6 (2004)) and Lee & Paull (Science 308, 551-4 (2005)).

Nuclease assay

[0186] The nuclease activity of MREIl was assayed as described by Paull & Gellert (MoI Cell 1,969-79 (1998)).

G2/M Checkpoint assay following Double Thymidine block

[0187] Cell Synchronization. U2OS cells were grown to about 40% confluency in 6- well plates. 24 hours after plating, 2 mM thymidine (Sigma) in DMEM was added to the cells for the first block. After 16 hours, thymidine was removed by washing the cells 2X with IX PBS. Fresh medium was added for 9 hours, then 2 mM thymidine was added for the second block for 16 hours. Thymidine was then removed followed by 2 washes with IX PBS, and fresh medium was added.

[0188] G2/M checkpoint assay. 8.5 hours following the second release, 25 μM Mirin, 10 μM Ku-55933 (ATM inhibitor) or DMSO was added to the cells. Thirty minutes later (9 hours post-release), cells were irradiated with 10 Gy of γ-radiation from a ¹³⁷Cs source or mock- treated, and incubated at 37°C for 1.5 hours. Each culture was then treated with 1 μg/mL nocodazole to trap cells in mitosis. Approximately 15 hours later, cells were harvested and fixed in 70% ethanol and placed at -20°C overnight. Cells were then resuspended in 1 ml of 0.25% Triton X-100 in PBS and incubated on ice for 15 min. After centrifugation, the cell pellet was suspended in 100 μl of 1% BSA-PBS with 1.5 μg of a polyclonal antibody directed against SerlO-phosphorylated form of histone H3 (Upstate Biotechnology). The cells were then rinsed with 1% BSA-PBS three times, incubated for 30 min with FITC-conjugated goat anti- rabbit 1 gG antibody (1:15 in 1% BSA-PBS, Jackson ImmunoResearch Laboratories, Inc.), rinsed three times with 1% BSA-PBS and stained with propidium iodide (Sigma) for 1 hour. Cellular fluorescence was then measured by flow cytometry. Homology-directed repair (HDR) assays

[0189] HDR assays were conducted essentially as described by Pierce et al. (Genes Dev 13, 2633-8 (1999)) using TOSA4, a subclone of 293T cells that contain a single integrated copy of the DR-GFP reporter. TOSA4 cells were seeded onto 60 mm plates at ~ 6 x 10⁵ cells/plate in 3 ml of non-selective medium. 24 hours later, the cells were transfected with 1 μg/plate of pCBASce, an expression vector encoding I-Scel, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. At 2 hours post-transfection, cells were treated with the indicated concentrations of Mirin (in DMSO) or with DMSO alone. At 24 hours after transfection, the cells were washed once with PBS and harvested. To measure homology- directed repair of an induced double- strand DNA break, the fraction of GFP-positive cells was quantified by flow cytometry. To evaluate cell cycle distribution, the cells were fixed in 70% ethanol at -2O°C, washed in 5 ml PBS containing 1% BSA, stained in 800 uL propidium iodide (Sigma) for 1 hour at room temperature, and analyzed on a FACScalibur flow cytometer.

Cytotoxicity assay

[0190] HEK293 cells were grown in DMEM with 10% fetal bovine serum and 1 % penicillin- streptomycin at 37°C in 5% CO₂. HEK293 cells were plated at approximately 500 cells per 100 mm plate and allowed to attach overnight (Day 0). On the following day, media was removed and 8 ml of fresh media supplemented with the appropriate concentration (0 μM, 10 μM, 25 μM, 50 μM, 100 μM) of Mirin was added to each plate (Day 1). Duplicate plates were set up for each dose. Cells were incubated with the compound for 24 hours at 37°C and 5% C02, after which media was aspirated and fresh, compound-free media was applied. Fresh media was applied every 3 days until Day 10. On Day 10 cells were fixed with 100% methanol and stained with 10% Giemsa for colony counting. Percentage survival is expressed as the average number of colonies on treated plate divided by average number of colonies on control plate (0 μM Mirin).

Mass Spectrometry

[0191] Mirin was analyzed in NBA matrix to determine purity using HXIlO Double- focusing Mass Spectrometer (JEOL Ltd., Tokyo, Japan). Resolution: 10,000. FAB ionization QkV Xe beam).

Preparation of Mirin

[0192] Referring now to Fig. 9; 4-hydroxybenzaldehyde (1.6 equivalents) was dissolved in glacial acetic acid (0.75 ml per mmole), and the resulting mixture was stirred to homogeneity. 2-Iminothiazolidin-4-one (Sigma- Aldrich, St. Louis, MO) was then added in one portion. Sodium acetate (4.2 equivalents) was added and the temperature was brought to reflux using a pre-heated oil bath. The initially homogeneous solution gave place to an orange red- precipitate. After 3.5 hours (or when thin layer chromatography indicated the consumption of starting material) the oil bath was removed, and the mixture was cooled to 4°C. The solids were filtered over Buchner, washed with water and then petroleum ether, and dried on high vacuum overnight. Z-5-(4-Hydroxyphenylidene)-2-imino-1,3-thiazolidin-4-one thus isolated displayed a purity greater than 95%. We have synthesized up to 22 grams of Mirin using this procedure, in approximately 50% yield from the first filtration crop.

Example 2: An inhibitor of the Mrell-RAD50-nbsl complex

[0193] To identify small molecules with potential radio- and/or chemo- sensitizing properties that inhibit the MRN-ATM pathway in response to DSBs a screen was used as described by Kasten et al. (Nature 432, 316-23 (2004)) and Zhou & Bartek (Nat Rev Cancer 4, 216-25 (2004)). The screen was performed in cell-free extracts derived from Xenopus eggs that recapitulate many aspects of the MRN- and ATM-dependent responses to DSBs and some of the aberrant phenotypes, such as radio-resistant DNA synthesis (RDS), observed in cancer prone syndromes (Dupre et al., Nat Struct MoI Biol 13,451-7 (2006); Costanzo et al., MoI Cell 6,649-59 (2000); Costanzo et al., MoI Cell 8, 137-47 (2001)). Xenopus extracts have been used previously to identify small molecule inhibitors of actin assembly, spindle assembly, and cell cycle progression (Peterson et al. Proc Natl Acad Sci U S A 98, 10624-9 (2001); Verma et el. Science 308, 117-20 (2004); Wignall et al., Chem Biol 11, 135-46 (2004)).

[0194] In the screen employed for the experiments described herein, the DNA damage response was triggered by incubating cell-free extracts with Haelll-digested plasmid DNA that mimics DNA DSBs. The read-out assay for ATM activity was phosphorylation of a peptide derived from histone H2AX (Costanzo et al., MoI Cell 6, 649-59 (2000); Costanzo et al., PLos Biol. 2, EIlO (2004)), carried out in a 96-well plate format (see FIG. IA and Example 1). In this forward chemical genetic screen, a small molecule could potentially target any step in the pathway leading to H2AX phosphorylation.

[0195] Agents screened for inhibition of H2AX phosphorylation (and hence are ATM inhibitors) included those in a 10,000-compound "DiverSet" library from Chembridge Corporation that had previously led to the identification of small molecules that inhibit p53 activity or interfere with mitosis and spindle dynamics (Komarov et al., Science 285, 1733-7 (1999); Mayer et al., Science 286, 971-4 (1999)). The robustness of the assay was confirmed by calculating the C value for each plate of the library (Zhang et al., J Biomol Screen 4, 67-73 (1999)). The average Z' value for the assay was 0.57 (FIG. IB). [0196] Using the above-described methods, the agent Z-5-(4-hydroxyphenylidene)-2- imino-1,3-thiazolidin-4-one (Mirin) was identified as an inhibitor of DSB-induced ATM activation (FIG: 1C). Purity of Mirin was confirmed by mass spectrometry (data not shown). The IC₅₀ for inhibition of H2AX phosphorylation by Mirin was estimated to be 65 μM (FIG. ID). A similar compound, Z-5-(4-hydroxybenzylidene)-2-thioxo-1,3-imidazolidin-4-one (FIG. 1C) did not inhibit DSBs- induced ATM activation (data not shown). Another compound (6- phenyl-2-thioxo-2,3- dihydro-4(1H)-pyrimidinone (compound IV), FIG. 1C) did not inhibit DSBs-induced ATM activation either, as seen by H2AX peptide phosphorylation (FIG. ID). To confirm the specificity of the assay for ATM kinase activity, ATM was immuno-depleted from extracts prior to addition of DSB-containing DNA and Mirin. H2AX phosphorylation was significantly reduced in ATM-depleted extracts and the residual H2AX phosphorylation was insensitive to Mirin, demonstrating that the inhibitory effect of Mirin on H2AX phosphorylation was entirely ATM-dependent (FIG. IE). Mirin also inhibited the ATM-dependent phosphorylation of the downstream targets NBSl and Chk2 (FIG. 2A) and the MRN-dependent autophosphorylation of ATM at S1981 in response to DSBs (FIG. 2B, lanes 3 and 5; FIG. 3C). This establishes Xenopus cell-free extracts as a valid and powerful system to identify small molecules that modulate the DNA damage response.

[0197] To identify the target of Mirin, extracts were depleted of the MRN complex using an antibody specific for MREIl and incubated with two different amounts of DSB- containing DNA corresponding to 1.2xlOⁿ ends/μl and 3.6xlO^π ends/μl respectively (FIG. 2B). Biotinylated DNA was generated by PCR using one biotinylated primer, purified and bound to streptavidin-bound as described by Dupre et al., (Nat Struct MoI Biol 13, 451-7 (2006)). After removal of streptavidin-bound DNA, ATM activation was monitored in the resulting soluble fractions (FIG. 2B). H2AX peptide was phosphorylated to similar extents in mock-depleted extracts at both DNA concentrations (FIG. 2B, lanes 2 and 4). MRN depletion prevented ATM activation at 1.2xlOⁿ ends/μl, but partial ATM activation was observed at 3.6xlO^π ends/μl (FIG. 2B, compare lanes 6 and 8). This confirms that MRN's requirement for ATM activation can be partially bypassed when the number of DNA breaks is increased, in agreement with results in mammalian NBS cells 30 and our own previous data Dupre et al., (Nat Struct MoI Biol 13, 451-7 (2006)). Notably, Mirin partially inhibited MRN-dependent ATM activation in mock-depleted extracts (FIG. 2B, lanes 3 and 5). In contrast, Mirin had no effect on MRN- independent ATM activity triggered by 3.6xlO^π ends/μl (FIG. 2B, lanes 7 and 9). This result strongly suggests that Mirin inhibits MRN-dependent activation of ATM kinase but not ATM kinase activity. [0198] To confirm this finding an in vitro assay was used with purified recombinant proteins and DNA. ATM was purified from mammalian cells as a catalytically inactive dimer (FIG. 2C; Lee & Paull, Science 308, 551-4 (2005)) or as a partly active monomer (FIG. 2D; Lee & Paull, Science 304, 93-6 (2004)), as previously described (Lee & Paull, Methods Enzymol 408, 529-39 (2006)). Activation of dimeric ATM by MRN and DNA was monitored using phosphorylation of recombinant p53 as a read-out with a phospho-specific antibody directed against Serl56. ATM activation was inhibited by Mirin with an IC₅₀ of 15 μM (FIG. 2C and data not shown). In contrast, the catalytic activity of monomeric ATM, which does not require MRN or DNA (Lee & Paull, Science 308, 551-4 (2005)), was not inhibited by 50 μM Mirin (FIG. 2D). Taken together, these data establish unambiguously that Mirin inhibits MRN- dependent activation of ATM by specifically targeting the MRN complex.

[0199] Next, tests were performed to ascertain if Mirin triggered MRN complex dissociation. Extracts were incubated with recombinant tagged complex

(MREl 1/RAD50/FLAG-NBS1) with or without 100 μM Mirin. MRN complexes were immunoprecipitated with FLAG antibodies. The complexes and the resultant soluble fractions were then analyzed by Western blot for the presence of MREIl, RAD50 and FLAG-NBSl (FIG. 3A). Complexes purified from Mirin-treated extracts were not dissociated and we did not observe the release of MRN subunits in soluble fractions (FIG. 3A). To test the possibility that Mirin could modulate the association of MRN with other proteins, extracts, treated or not with 100 μM Mirin, were subjected to Superose-6 chromatography (Dupre et al., Nat Struct MoI Biol 13, 451-7 (2006)) and the resulting fractions probed for MREIl (FIG. 3B). The elution profile of MREIl -containing fractions was not affected by Mirin, demonstrating that endogenous MREIl -containing complexes were not dissociated by Mirin.

[0200] The MRN complex exhibits DNA-binding and DNA-tethering activities, both of which are required for ATM activation (You et al., MoI Cell Biol 25, 5363-79 (2005); Dupre et al. Nat Struct MoI Biol 13, 451-7 (2006); Falck et al., Nature 434, 605-11 (2005); Costanzo et al., PLoS Biol 2, EIlO (2004)). MRN promotes ATM recruitment to damaged DNA(You et al., MoI Cell Biol 25, 5363-79 (2005); Dupre et al. Nat Struct MoI Biol 13, 451-7 (2006); Falck et al., Nature 434, 605-11 (2005)). Therefore, further tests were performed to determine if MRN promotion of ATM recruitment to damaged DNA is inhibited by Mirin. In particular, the association of ATM and MREIl with DNA was monitored by Western blot analysis of the DNA-bound fractions isolated from extracts incubated with streptavidin-bound biotinylated DNA (FIG. 3C). At concentrations up to 1 mM, Mirin did not affect ATM or MREIl binding to DNA (FIG. 3C). [0201] Next, the MRN-mediated DNA-tethering activity of cell-free extracts was measured in the presence of Mirin. It had previously been shown that 50% of DNA tethering observed in cell-free extracts is MRN dependent (Dupre et al. Nat Struct MoI Biol 13, 451-7 (2006)). DNA tethering was assessed by monitoring the ability of biotinylated DNA bound to streptavidin to associate with non-biotinylated, radioactive DNA (FIG. 3d). Mirin did not inhibit DNA tethering at 10 μM Mirin (FIG. 3D). In fact, 100 μM Mirin reproducibly stimulated MRN-dependent DNA tethering (FIG. 3D). This could reflect a slow turnover of MRN complex on DNA in the presence of Mirin. As anticipated, MREl 1 depletion reduced DNA tethering by 50%, and the residual, MRN-independent, DNA-tethering activity was not affected by Mirin (FIG. 3D). Recently it was shown that RAD50-associated adenylate kinase activity was required for MRN-dependent DNA tethering (Venugopal Bhaskara et al. Molecular cell, 25(5), 647-61 (2007)). Nevertheless, this activity was not inhibited by Mirin (data not shown). Finally, tests were performed to ascertain whether Mirin affects the 3' to 5' exonuclease activity of MRN 17 by monitoring the appearance of discretely-digested DNA products on polyacrylamide gels (FIG. 3E). Mirin inhibited MREIl nuclease activity at 100 μM (FIG. 3E, lane 5), but did not inhibit ExoIII nuclease activity (data not shown). This result confirms that MREIl is a direct target for Mirin, consistent with the experiments in cell- free extracts (FIG. 2B) and the in vitro experiments using recombinant proteins (Fig. 2c). Taken together, these data identify Mirin as a novel MREIl inhibitor, and also suggest that the nuclease and DNA end-processing functions of MREIl are required for proper ATM activation.

[0202] Next experiments were performed to determine the consequences of exposing mammalian cells to Mirin. Exponentially growing TOSA4 (FIG. 4A) and U2OS (FIG. 4B) cells were treated with increasing concentrations of Mirin (TOS A4). Mirin triggered a significant G2/M arrest at concentrations of 50 μM and 100 μM. This observation suggests that Mirin elicits a DNA damage checkpoint in G2, consistent with the role for MRN in the maintenance of genome stability during S-phase (Costanzo et al., PLoS Biol 2, EIlO (2004); Trenz et al., EMBO J 25, 1764-74 (2006)). To investigate the nature of this arrest, U2OS cells were treated with 100 μM Mirin or with 100 μM Mirin and 10 μM of KU-55933, a specific ATM inhibitor (Hickson et al., Cancer Res 64, 9152-9 (2004)). A similar G2 arrest was observed under these conditions (FIG. 4B) establishing that this G2/M arrest was ATM independent. However, an inhibitor of the MRN-ATM pathway is predicted to abolish the IR- induced G2/M checkpoint. To circumvent Mirin-induced G2/M arrest observed in asynchronously growing cells, we assessed the consequences of exposing G2 cells to Mirin. U2OS cells were synchronized in Gl by double thymidine block. Following release, cells progressed synchronously through S-phase and reached G2 at 8.5 hrs (data not shown). G2 cells were treated with 100 μM Mirin, irradiated with 10 Gy, treated with nocodazole and stained for phosphorylated histone H3, a marker of mitotic cells. Treatment with Mirin resulted in a 10-fold increase in cells entering mitosis following IR (FIG. 4D). This establishes that Mirin can abolish the IR-induced G2/M checkpoint, consistent with its ability to inhibit ATM signaling.

[0203] Next, the effects of Mirin on DNA repair in mammalian cells were examined. We first analyzed the cellular cytotoxicity of Mirin (FIG. 5A). HEK293 cells were exposed to increasing concentrations of Mirin for 24 hours and cell survival was analyzed 10 days later. Mirin had little effect on cell survival at 25 μM and showed 50% cytotoxicity at 50 μM. The effect of Mirin on homology-directed repair (HDR) of double- strand DNA breaks (DSBs) in HEK293-derived, TOSA4 cells that harbor a single chromosomally-integrated copy of the DR- GFP reporter substrate was investigated next. The DR-GFP reporter harbors two nonfunctional copies of the GFP gene, one (SceGFP) that is disrupted by the recognition site for the rare-cutting endonuclease I-Scel and another (iGFP) that only encodes an internal region of the GFP polypeptide (FIG. 5B) (Pierce et al., Genes Dev. 13: 2633-38 (1999); Esashi et al., Nature 434: 598-604 (2005)). Expression of I-Scel in TOSA4 cells results in a chromosomal DSB at the I-Scel site of SceGFP, and repair of the induced DSB by gene conversion with iGFP yields cells expressing a functional GFP⁺ gene that can be scored by flow cytometry. Treating these cells for 24 hrs with 25 μM of Mirin, a dose that does not affect cell survival significantly (FIG. 5A), decreased the appearance of GFP positive cells by 70% (FIG. 5B). This result demonstrates that Mirin inhibits HDR and supports the idea that MRN, and possibly the nuclease activity of MREIl, are essential for HDR in mammalian cells. Inhibition of HDR by Mirin was not a consequence of cell cycle arrest in Gl (FIG. 4A).

[0204] Thus, the results described above demonstrate the Mirin is a new inhibitor of the MRN complex that specifically targets MREIl nuclease activity. As shown, Mirin is a powerful tool that can rapidly inhibit MRN activity in mammalian cells thus circumventing the lethality associated with genetic inactivation of the complex. In addition, the results indicate that MREIl nuclease activity and subsequent DNA end processing are required to activate ATM and to promote HDR.

Example 3: Identifying Cancer/Tumor Types Sensitive to the Present Inhibitors [0205] This Example describes tests that can be performed to determine which cancer cell types are most responsive to inhibition of homology-dependent repair of DNA.

[0206] Cell lines. Diverse types of cancer cells and tissue samples can be screened. Initial work has focused on testing the radiosensitizing potential of Mirin on U2OS cells because they are easily synchronized, have very well characterized checkpoint and have been used for siRNA studies. Other cell types that can be tested include MCF7, MDA-MB-231, HS 578T, and HCT-116 (mismatch repair deficient) because a wealth of information has been accumulated by the NCI about screening small compounds with these cell lines. Another cell type that can be tested is GM08437 cells, which are deficient in nucleotide excision repair and that can be complemented with XPF. In addition, a collection of human cancer cell lines from the ATCC can be tested, for example, ATCC cancer cells that can be obtained with the normal cell lines derived from the same patients

(http://www.atcc.org/common/cultures/NavByApp.cfm). For example, a small cell lung cancer cell line, a malignant melanoma cell line and an osteosarcoma cell line along with normal peripheral blood cell lines from the same patients can be tested.

[0207] siRNAs. To down-regulate the MRN complex the following siRNA can be used: MREIl:

ACAGGAGAAGAGATCAACT (SEQ ID NO:6);

GCTAATGACTCTGATGATA (SEQ ID NO: 18); RAD50:

GGAGAAGGAAATACCAGAA (SEQ ID NO: 12),

GCTCAGAGATTGTGAAATG (SEQ ID NO: 19); NBSl:

CAGGAGGAAGATGTCAATG (SEQ ID NO: 15);

GAAGAAACGTGAACTCAAG (SEQ ID NO: 16)

See, Chai et al., EMBO Rep 7, 225 (Feb, 2006); Myers et al., / Biol Chem 281, 9346 (Apr 7, 2006)).

[0208] Cytotoxicity assay. The rapid luminescent CellTiter-Glo assay from Promega can be used.

[0209] Methods. Each cell line will be treated with 25 μM and 100 μM of Mirin for 24 hours and cell viability will be assessed using a rapid luminescent assay. If significant cytotoxicity is observed with some cell lines, a titration of Mirin will be performed. The matched control cell lines will be tested similarly, when available. Furthermore, the sensitivity of these cells to MRN inhibition will be validated using siRNA against MREIl, RAD50, and NBSl. Treatment with siRNAs will be performed according to standard protocols.

[0210] Next tests can be performed to ascertain if MREIl inhibition with Mirin sensitizes cells to killing by irradiation. First, the radiosensitivity of each cell line will be determined using doses ranging from 0.1 -20 Grays. For each cell line, a dose that triggers modest cytotoxicity will be chosen, for example, in the range of 20%. This pre-determined dose of irradiation will be combined with treatment with Mirin at a dose that results in modest cytotoxicity in isolation, as established in the first set of experiments. If synergistic killing is observed in cells treated with Mirin and irradiation, the Mirin concentration and the dose of irradiation will be we will titrated to determine the optimal conditions for cell killing.

[0211] Treatment with Mirin can also be combined with PARP inhibition using inhibitors that are currently undergoing clinical trials, for example, those described in Plummer, Cur Opin Phamacol 6, 364 (Aug, 2006), which is incorporated by reference in its entirety.

[0212] Preliminary experiments strongly support the idea that treating HDR-deficient tumor cells with PARP inhibitors will be a successful to treat BRCAl^-7- and BRCA 2^~'^~ tumors. This can be expanded to the treatment of HDR-proficient cancer cells, which represent the majority of cancers. This can permit single agent therapy for tumors with repair deficiencies and dual agent therapy for other tumors.

[0213] The documents, including any patent document, cited herein, including the documents identified below are hereby incorporated by reference as if recited in full.

Cited Documents

1. Zhou, B. B. & Elledge, S. J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433-9 (2000).

2. Kastan, M. B. & Bartek, J. Cell-cycle checkpoints and cancer. Nature 432, 316-23 (2004).

3. Lavin, M. .F. et al. ATM signaling and genomic stability in response to DNA damage. Mutat Res 569, 123-32 (2005).

4. You, Z., Chahwan, C, Bailis, J., Hunter, T. & Russell, P. ATM activation and its recruitment to damaged DNA require binding to the C terminus of NBSl. MoI Cell Biol 25, 5363-79 (2005).

5. Lee, J. H. & Paull, T. T. Direct activation of the ATM protein kinase by the Mrell/Rad50/Nbsl complex. Science 304, 93-6 (2004).

6. Lee, J. H. & Paull, T. T. ATM activation by DNA double-strand breaks through the Mrell-Rad50-nbsl complex. Science 308, 551-4 (2005). 7. Carson, C. T. et al. The Mrell complex is required for ATM activation and the G2/M checkpoint. Embo J 22, 6610-20 (2003).

8. Difilippantonio, S. et al. Role of NBSl in the activation of the Atm kinase revealed in humanized mouse models. Nat Cell Biol 7, 675-85 (2005).

9. Dupre, A., Boyer-Chatenet, L. & Gautier, J. Two-step activation of ATM by DNA and the Mrell -Rad50-Nbsl complex. Nat Struct MoI Biol 13, 451-7 (2006).

10. Kozlov, S. V. et al. Involvement of novel autophosphorylation sites in ATM activation. Embo J 25, 3504-14 (2006).

11. Gatei, M. et al. ATM-dependent phosphorylation of nibrin in response to radiation exposure. Nat Genet 25, 115-9 (2000).

12. Paull, T. T. & Lee, J. H. The Mrell/Rad50/Nbsl complex and its role as a DNA double-strand break sensor for ATM. Cell Cycle 4, 737-40 (2005).

13. Falck, J. Coates, J. & Jackson, S. P. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature 434, 605-11(2005).

14. Uziel, T. et al. Requirement of the MRN complex for ATM activation by DNA damage. Embo J 22, 5612-21 (2003).

15. Shiloh, Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer 3, 155-68 (2003).

16. D'Amours, D. & Jackson, S. P. The Mrell complex: at the crossroads of dna repair and checkpoint signaling. Nat Rev MoI Cell Biol 3, 317-27 (2002).

17. Paull, T. T. & Gellert, M. The 3' to 5' exonuclease activity of Mrell facilitates repair of DNA double-strand breaks. MoI Cell 1, 969-79 (1998).

18. Haber, J. E. The many interfaces of Mrell. Cell 95, 583-6 (1998).

19. Zhou, B. B. & Bartek, J. Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat Rev Cancer 4, 216-25 (2004).

20. Costanzo, V. et al. Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage. MoI Cell 6, 649-59 (2000).

21. Costanzo, V. et al. Mrell protein complex prevents double-strand break accumulation during chromosomal DNA replication. MoI Cell 8, 137-47 (2001).

22. Peterson, J. R., Lokey, R. S., Mitchison, T. J. & Kirschner, M. W. A chemical inhibitor of N-WASP reveals a new mechanism for targeting protein interactions. Proc Natl Acad Sci USA 98, 10624-9 (2001).

23. Verma, R. et al. Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain. Science 306, 117-20 (2004). 24. Wignall, S. M. et al. Identification of a novel protein regulating microtubule stability through a chemical approach. Chem Biol 11, 135-46 (2004).

25. Costanzo, V., Paull, T., Gottesman, M. & Gautier, J. Mrell assembles linear DNA fragments into DNA damage signaling complexes. PLoS Biol 2, EIlO (2004).

26. Komarov, P. G. et al. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy. Science 285, 1733-7 (1999).

27. Mayer, T. U. et al. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971-4 (1999).

28. Zhang, J. H., Chung, T. D. & Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73 (1999).

29. Cimprich, K. A., Shin, T. B., Keith, C. T. & Schreiber, S. L. cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein. Proc Natl Acad Sci USA 93, 2850-5 (1996).

30. Kitagawa, R., Bakkenist, C. J., McKinnon, P. J. & Kastan, M. B. Phosphorylation of SMCl is a critical downstream event in the ATM-NBS1-BRCA1 pathway. Genes Dev 18, 1423-38 (2004).

31. Lee, J. H. & Paull, T. T. Purification and biochemical characterization of ataxia- telangiectasia mutated and Mrell/Rad50/Nbsl. Methods Enzymol 408, 529-39 (2006).

32. Venugopal Bhaskara, A. D., Ben Hopkins, Bettina Lengsfeld, & Ji-Hoon Lee, X. Z., Jean Gautier, Virginia Zakian, and Tanya T. Paull. Rad50 Adenylate Kinase Activity Regulates DNA tethering activities of Mrell/Rad50 complexes. Molecular cell, 25(5), 647-61 (2007).

33. Trenz, K., Smith, E., Smith, S. & Costanzo, V. ATM and ATR promote Mrell dependent restart of collapsed replication forks and prevent accumulation of DNA breaks. Embo J 25, 1764-74 (2006).

34. Hickson, I. et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res 64, 9152- 9 (2004).

35. Pierce, A. J., Johnson, R. D., Thompson, L. H. & Jasin, M. XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13, 2633-8 (1999).

36. Esashi, F. et al. CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair. Nature 434, 598-604 (2005).

37. Smythe, C. & Newport, J. W. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell Biol 35,449- 88 (1991). 38. E. R. Plummer, Curr Opin Pharmacol 6, 364 (Aug 2006).

39. W. Chai, A. J. Sfeir, H. Hoshiyama, J. W. Shay, W. E. Wright, EMBO Rep 7, 225 (Feb, 2006).

40. J. S. Myers, D. Cortez, J Biol Chem 281, 9346 (Apr 7, 2006).

41. H. Farmer et al., Nature 434, 917 (Apr 14, 2005).

42. D. Domingues-Sola et al., Nature In press (2007).

43. J. Bartkova et al., Nature 434, 864 (Apr 14, 2005).

44. H. E. Bryant et al., Nature 434, 913 (Apr 14, 2005).

[0214] The terms and expressions that have been employed herein are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0215] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Claims

What is claimed is:

1. A composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I:

wherein:

L is a bond or a linker selected from C₁-C₄ alkylene, C₁-C₄ alkenylene, or C₂-C₄ alkynylene;

B is selected from the group consisting of phenyl, ortho-fused bicyclic carbocyclic, and heteroaryl, wherein the ortho-fused bicyclic carbocyclic has nine or ten ring atoms and in which at least one ring is aromatic, and wherein the heteroaryl is a radical attached to L via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and from one to four Y_n where n is 1 or a radical attached to A via a ring carbon of an ortho-fused bicyclic heterocycle consisting of eight to ten ring atoms wherein one of the rings is an aromatic ring containing five or six ring atoms consisting of carbon and from one to four Y_n where n=l;

2. The composition of claim 1, wherein the compound has formula II:

wherein X, Y and Z are as defined in claim 1.

3. The composition of any of claims 1 or 2, wherein the compound has the structure:

4. The composition of any of claims 1-3, further comprising a chemotherapeutic agent.

5. A method of treating cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula I:

wherein:

Y is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(Q), wherein Q is absent or is H, O, (C,-CJaIlCyI, phenyl, or benzyl;

6. The method of claim 5, wherein the compound has formula II:

wherein X, Y and Z are as defined in claim 5.

7. The method of any of claims 5 or 6, wherein the compound has the structure:

8. The method of any of claims 5, 6 or 7, wherein the compound is administered with a chemotherapeutic agent.

9. The method of claim 8, wherein the chemotherapeutic agent is an anti- microtubule agent, platinum coordination complex, alkylating agent, antibiotic, topoisomerase II inhibitor, antimetabolite, topoisomerase I inhibitor, hormone, signal transduction pathway inhibitor, non-receptor tyrosine kinase angiogenesis inhibitor, immunotherapeutic agent, proapoptotic agent, cell cycle signaling inhibitor, or a combination thereof.

10. The method of claim 8, wherein the chemotherapeutic agent is altretamine, aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, calcium folinate, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, crisantaspase, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nirutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, vinorelbine, tamoxifen, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-α- morpholinyl)propoxy)-quinazoline, 4-(3-ethynylphenylamino)-6,7-bis(2- methoxyethoxy)quinazoline, hormones, steroids, 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminogrutethimide, estramustine, medroxyprogesteroneacetate, leuprolide, flutamide, toremifene, Zoladex, antiangiogenics, matrix metalloproteinase inhibitors, VEGF inhibitors, ZD6474, SU6668, SUl 1248, anti-Her-2 antibody (e.g., ZD1839 and OS1774), EGFR inhibitor, EKB-569, Imclone antibody C225, src inhibitor, bicalutamide, epidermal growth factor inhibitors, Her-2 inhibitor, MEK-I kinase inhibitor, MAPK kinase inhibitor, Pl 3 inhibitor, PDGF inhibitor, combretastatin, MET kinase inhibitor, MAP kinase inhibitors, inhibitor of non-receptor and receptor tyrosine kinases (imatinib), inhibitor of integrin signaling, inhibitor of insulin-like growth factor receptors, an EGF-receptor antagonist, arsenic sulfide, adriamycin, cisplatin, carboplatin, cimetidine, carminomycin, mechlorethamine hydrochloride, pentamethylmelamine, thiotepa, teniposide, cyclophosphamide, chlorambucil, demethoxyhypocrellin A, melphalan, ifosfamide, trofosfamide, Treosulfan, podophyllotoxin, etoposide phosphate, teniposide, etoposide, leurosidine, leurosine, vindesine, 9-aminocamptothecin, camptoirinotecan, crisnatol, Chloroambucil, megestrol, methopterin, mitomycin C, ecteinascidin 743, busulfan, carmustine (BCNU), lomustine (CCNU), lovastatin, l-methyl-4-phenylpyridinium ion, semustine, staurosporine, streptozocin, thiotepa, phthalocyanine, dacarbazine, aminopterin, methotrexate, trimetrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin, cytarabine (ara C), porfiromycin, 5-fluorouracil, 6-mercaptopurine, doxorubicin hydrochloride, leucovorin, mycophenoloc acid, daunorubicin, deferoxamine, floxuridine, doxifluridine, ratitrexed, idarubicin, epirubican, pirarubican, zorubicin, mitoxantrone, bleomycin sulfate, mitomycin C, actinomycin D, safracins, saframycins, quinocarcins, discodermolides, vincristine, vinblastine, vinorelbine tartrate, vertoporfin, paclitaxel, tamoxifen, raloxifene, tiazofuran, thioguanine, ribavirin, EICAR, estramustine, estramustine phosphate sodium, flutamide, bicalutamide, buserelin, leuprolide, pteridines, diyneses, levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride, betamethosone, gemcitabine hydrochloride, verapamil, VP- 16, altretamine, thapsigargin, topotecan and combinations thereof.

11. A method of inhibiting MREl 1 activity comprising administering to a mammal a therapeutically effective amount of a compound of formula I:

wherein:

L is a bond or a linker selected from C₁-C₄alkylene, C₁-C₄ alkenylene, or C₂-C₄ alkynylene;

Y is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(Q), wherein Q is absent or is H, O, (C₁-CJaIkVl, phenyl, or benzyl;

12. A method of sensitizing cancer cells to chemotherapy or radiation therapy comprising administering to a mammal a therapeutically effective amount of a compound of formula I:

wherein:

13. A method of identifying an anticancer agent comprising: a. contacting an assay mixture with a test agent, wherein the assay mixture compiles a H2AX peptide having the amino acid sequence of SEQ ID NO:2, ATP, DNA with double- stranded breaks, and an inhibitor of MREl 1, RAD50, NBSl, or a combination thereof; b. observing whether phosphorylation of the peptide (SEQ ID NO:2) increases relative to a control assay lacking the test agent; and c. identifying the test agent as an anticancer agent when the phosphorylation of the peptide (SEQ ID NO:2) increases.

14. A method of identifying an agent that can modulate a pathway involving MREIl, RAD50, NBSl, and/or ATM comprising: a. contacting an assay mixture with a test agent, wherein the assay mixture comprises a H2AX peptide having the amino acid sequence of SEQ ID NO:2, ATP, DNA with double- stranded breaks, and an inhibitor of MREl 1, RAD50, NBSl, or a combination thereof; b. determining whether phosphorylation of the polypeptide (SEQ ID NO:2) increases or decreases relative to a control assay that does not contain the test agent; and c. identifying an agent that modulates the MRN-ATM pathway when that agent increases or decreases the phosphorylation of the polypeptide (SEQ ID NO:2) relative to the control assay.

15. The method of claim 13 or 14, wherein the assay mixture further comprises a cytoplasmic extract and calcium.

16. The method of claim 15, wherein the cytoplasmic extract is a Xenopus oocyte cytoplasmic extract.

17. The method of claim 13 or 14, wherein the ATP has a detectable label on its terminal (γ) phosphate.

18. The method of claim 13 or 14, wherein each assay is performed in a well of a 96- well plate with V-shaped wells.

19. The method of claim 18, wherein each assay is performed in an assay volume of about 1-5 microliters.

20. The method of claim 13 or 14, wherein the assay mixtures are transferred to P81 phosphocellulose coated 96-well plates.

21. The method of claim 20, wherein the peptide (SEQ ID NO:2) binds to the p81 pho sphocellulo se .

22. The method of claim 21, wherein phosphorylation of the peptide (SEQ ID NO:2) is detected by detecting a labeled phosphate linked to the peptide.

23. The method of claim 21, wherein phosphorylation of the H2AX (SEQ ID NO:2) peptide is detected using an antibody that detects phosphorylated H2AX (SEQ ID NO:2) peptide but substantially no non-phosphorylated H2AX (SEQ ID NO:2) peptide.

24. A method of identifying an anticancer agent comprising: a. contacting an assay mixture with a test agent, wherein the assay mixture comprises cancer cells and an inhibitor of MREIl, RAD50, NBSl, or a combination thereof; b. observing whether growth of the cancer cells in step (a) changes; and c. identifying the test agent as an anticancer agent if the cancer cell growth decreases.

25. The method of claim 13 or 23, wherein the inhibitor is a compound of formula I:

wherein:

B is selected from the group consisting of phenyl, ortho-fused bicyclic carbocyclic, and heteroaryl, wherein the ortho-fused bicyclic carbocyclic has nine or ten ring atoms and in which at least one ring is aromatic, and wherein the heteroaryl is a radical attached to L via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and from one to four Y_n where n is 1, or a radical attached to A via a ring carbon of an ortho-fused bicyclic heterocycle consisting of eight to ten ring atoms wherein one of the rings is an aromatic ring containing five or six ring atoms consisting of carbon and from one to four Y_n where n=l; X is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(P),wherein P is absent or is H;

Y is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(Q), wherein Q is absent or is H, O, (C₁-CJaIlCyI, phenyl, or benzyl;

26. The method of claim 13 or 23, wherein the inhibitor is a nucleic acid that inhibits the expression or translation of MREIl, RAD50, NBSl, or a combination thereof.

27. The method of claim 25, wherein the nucleic acid is homologous or complementary to about 10 to about 50 nucleotides of SEQ ID NO:3, 4, or 5.

28. A method of inhibiting MRN (MREl l-RAD50-NBSl)-dependent activation of ATM (Ataxia- Telangiectasia Mutated) comprising contacting a cell capable of expressing the MRN (MREl 1-RAD50-NBS1) with at least one nucleic acid that inhibits the expression or translation of MREIl, RAD50, NBSl, or a combination thereof.

29. The method of claim 27, wherein the nucleic acid is about 10-50 nucleotides in length, or has any number of nucleotides from about 10 to about 50 nucleotides.

30. The method of claim 27 or 28, wherein the nucleic acid is homologous or complementary to about 10 to about 50 nucleotides of SEQ ID NO:3, 4, or 5.

31. The method of any of claims 27-29, wherein the nucleic acid consists of any one of SEQ ID NO:6-17, or a combination thereof.

32. The method of any of claims 27-30, further comprising contacting the cell with a compound of formula I:

Y is a heteroatom or substituted heteroatom selected from the group consisting of non- peroxide oxygen, sulfur, and N(Q)₁ wherein Q is absent or is H, O, (C₁-C₄)alkyl, phenyl, or benzyl;

33. The method of any of claims 27-31, wherein the cell is within a mammal.

34. The method of claim 32, further comprising administering to the mammal a chemotherapeutic agent.

35. A method of inhibiting MREIl activity comprising contacting the MREIl with a compound of the following structure:

to thereby inhibit MREl 1 activity

36. A method of inhibiting MRN (MREl l-RAD50-NBSl)-dependent activation of ATM (Ataxia- Telangiectasia Mutated) comprising contacting MRN with a compound of the following structure:

to thereby inhibit MRN-dependent activation of ATM.

37. The method of claim 35 or 36, wherein the method is performed in vitro.

38. The method of claim 35 or 36, wherein the method is performed in a living cell.